JOURNAL

OF

BACTERIOLOGY, Jan. 1991, p. 791-800

Vol. 173, No. 2

0021-9193/91/020791-10$02.00/0 Copyright C 1991, American Society for Microbiology

Sodium Ion-Dependent Amino Acid Transport in Membrane Vesicles of Bacillus stearothermophilus RENE I. R. HEYNE, WIM DE VRIJ, WIM CRIELAARD, AND WIL N. KONINGS* Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Received 27 August 1990/Accepted 9 November 1990

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (K, = 0.4 mM). In contrast, the Na'-H+-L-glutamate transport system has a high affinity for sodium ions (Kt < 5.5 ,uM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial A* and AILNa+/F but not a ApH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allostericaily affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vm,.) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism. In bacteria, energy-consuming processes such as solute transport can utilize electrochemical proton gradients as a driving force. Although H+ is most often the central coupling ion in bacterial energy transduction (30), Na+ can often perform this function and connect endergonic with exergonic processes. Na+ ions are essential for growth of many marine, halophilic, alkalophilic, and rumen bacteria which live in Na+-rich habitats (4, 11, 28, 31) and are an important growth factor for methanogenic bacteria (29) and for some freshwater organisms when special substrates are utilized (11). Na+ plays a role not only in Na+-coupled energy transduction mechanisms (11) such as Na+-solute symport systems (9, 28, 31) but also in pH homeostasis (4, 26) and the activities of many enzymes (11). The advantage of using Na+-dependent transport systems is clear under specific culture conditions such as a high environmental pH or a high external sodium concentration. For example, in obligate alkalophilic bacilli, primary proton extrusion is followed by Na+-dependent proton accumulation, resulting in a reversed proton gradient ([H'in] > [H+,ut]) and an inwardly directed Na+ gradient ([Na+in] < [NaIOut]) (23). This electrogenic Na+/H+ antiport maintains a physiological cytoplasmic pH and supplies the bacteria with a chemical gradient of sodium ions. Several other marine or alkalophilic bacteria (possessing Na+-solute transport systems) can generate an electrochemical gradient of sodium ions with primary Na+ pumps such as Na+-pumping decarboxylases (11), ATP hydrolases (17), and redox-converting enzymes (30, 32). Almost all bacteria living under extreme pH conditions possess Na+dependent transport systems, but in many neutrophilic bacteria Na+-dependent transport systems have been recognized as well (5, 6, 9, 13, 14, 16, 28, 31, 34, 35). The advantage of having Na+-dependent instead of H+-depen*

dent transport systems in these latter organisms is not

always clear. Symport of both H+ and Na+ with the same solute has been observed for the L-glutamate transport systems of Escherichia coli (13, 14) and Bacillus stearothermophilus (9) and citrate transport systems in Klebsiella pneumoniae (11). Also, transport systems which can use either H+, Na+, or Li' as a coupling ion have been reported.

The melibiose transport system of E. coli can switch coupling ions, depending on the sugar solute used (3, 33). Similar characteristics have been reported for the alanine carrier of the thermophile PS3 (18-22), a bacterium strongly resembling B. stearothermophilus. Many Na+-solute symport systems have been reported. The major proline and serine/threonine transport systems of E. coli (6, 16) and the proline transport system of Salmonella typhimurium (5) use Na+ as a coupling ion. In contrast to these systems, which display a high affinity for Na+, several low-affinity Na+ symport transport systems (K, in the millimolar range) have recently been described for the rumen bacterium Streptococcus bovis (28) and the thermophilic fermentative bacterium Clostridium fervidus (31). In this report, we describe the mechanistic aspects of amino acid transport in membrane vesicles from the thermophilic bacterium B. stearothermophilus. The acidic amino acids L-glutamate and L-aspartate are translocated in symport with one Na+ ion and one H+ ion. In this study we also analyzed the mechanisms of neutral (branched-chain) and basic amino acid transport. For the neutral amino acids, Na+-dependent transport systems have been found. MATERIALS AND METHODS Cell growth and preparation of membrane vesicles. B.

stearothermophilus ATCC 7954 was grown at 63°C with vigorous aeration in a medium containing 2% (wt/vol) tryptone, 1% (wt/vol) yeast extract, and 1% (wt/vol) NaCl

Corresponding author. 791

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HEYNE ET AL.

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Time (min) Time (min) FIG. 1. Effect of ionophores on the uptake of L-alanine (A) and L-leucine (B) in membrane vesicles of B. stearothermophilus. Uptake of L-alanine (2.87 ,uM) or L-leucine (1.43 I1M) was measured at 45°C in oxygen-saturated 50 mM MES-NaOH (pH 6.0) containing 10 mM MgSO4. The figure shows L-alanine and L-leucine uptake in the absence of electron donors (0), in the presence of the electron donors ascorbate (20 mM) and TMPD (300 ,uM) without ionophores (0), and in the presence of monensin (20 nM) (L) or nonactin (10 ,uM) (E). Final protein concentration, 0.28 mg/ml.

adjusted to pH 7.0. Exponentially growing cells were harvested at an A6. of 1.5 to 2.0, washed once, and resuspended in 50 mM potassium phosphate (pH 7.0). Membrane vesicles were prepared essentially as described by Konings et al. (24), but with the modification that lysozyme treatment was performed at 50°C instead of 37°C to facilitate cell wall breakdown. Concentrated membranes (20 to 30 mg of protein per ml) were washed once and resuspended in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH (pH 6.0) and subsequently stored in liquid nitrogen. Transport assays. Amino acid uptake was assayed as described by de Vrij et al. (9) at 45 or 50°C. Membrane vesicles were suspended in an assay mixture containing 0.1 to 0.2 ml of oxygen-saturated buffer supplemented with 10 mM MgSO4 and 20 mM ascorbate of appropriate pH (see figure legends for details). After 1 min of incubation, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD; 100 to 300 puM) was added. Transport of amino acids driven by artificial gradients was performed as described by de Vrij et al. (9) at 45°C. For kinetic analysis of proton motive force (Ap)-driven uptake, the membrane vesicles were washed twice and resuspended in 50 mM Tris hydrochloride (pH 7.0). Uptake was performed at 50°C in the same buffer supplemented with 50 mM NaCl and 10 mM MgSO4 by measuring the amount of label accumulated during the first 10 s. The external concentration of 14C-labeled substrate was varied by the addition of unlabeled substrate. Results were analyzed with Eadie-Hofstee plots. Inhibitor constants (K,) were estimated by measuring the rate of amino acid uptake

in the absence (Jo) or presence (J,) of a 10- to 30-fold excess of inhibitor by using the following equation (12): Ki = [JI(J0 - J.)] (K, [I])/(K, + [SI) where K, is the affinity constant of the amino acid assayed and [I] and [S] are the inhibitor and solute concentrations, respectively. The cation dependency of the different amino acid transport systems was determined by measuring the initial rate of amino acid uptake as described above but by replacing NaCl with 50 mM chloride salts of one of the monovalent cations Li+, K+, Cs+, Rb+, and NH4+. Sodium contaminant concentrations under these conditions range from 5 to 6 p,M. The sodium dependency of transport was determined by measuring the initial uptake rate in the presence of different concentrations of NaCl (0 to 50 mM). Results were analyzed with Eadie-Hofstee plots. Determination of transmembrane gradients. Qualitative determinations of the membrane potential (A,&; interior negative) were performed by measuring the absorbance changes (A63 - A6.) of the membrane potential indicator 3,3'dipropylthiocarbocyanine iodide [diSC(3)5] (9). The assay mixtures contained membrane vesicles (0.03 to 0.1 mg of protein per ml), ascorbate (20 mM) and TMPD (100 ,uM) as an electron donor system, and diSC(3)5 (3 ,uM). The measurements were performed at 45°C in different oxygensaturated buffers. Monensin (20 nM), nonactin (10 ,M), and 3,5-di-tert-butyl-4-hydroxy-benzelidene mononitrile (SF6847; 100 nM) were used to dissipate the ApH, the A+, and the complete Ap, respectively. pH gradients across the -

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BACILLUS Na+-DEPENDENT AMINO ACID TRANSPORT

VOL. 173, 1991

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Time (min) FIG. 2. Effect of ionophores on the uptake of L-lysine in membrane vesicles of B. stearothermophilus. Uptake of L-lysine (1.54 ,uM) was measured as described in the legend to Fig. 1. The figure shows L-lysine uptake in the absence of electron donors (0), in the presence of the electron donors ascorbate (20 mM) and TMPD (300 ,M) without ionophores (0), and in the presence of monensin (20 nM) (U) or SF-6847 (1 pLM) (O). Final protein concentration, 0.23

mg/ml.

membrane (ApH; interior alkaline) were determined at 45°C from the fluorescence changes of pyranine (excitation at 460 nm and emission at 508 nm [7, 8]) entrapped in B. stearothermophilus membrane vesicles by using a Perkin-Elmer LS 50 luminescence spectrophotometer (Perkin-Elmer Corp., Beaconsfield, United Kingdom). Pyranine was entrapped within the intravesicular space by adding 200 ,uM pyranine to the membrane vesicles before freeze-thawing (12). External pyranine was removed by chromatography of the membrane suspension over a Sephadex G-25 column (coarse grade; 1 by 20 cm) preequilibrated with the appropriate buffer. ApH determinations were also performed in different air-saturated buffers of appropriate pH containing 10 mM MgSO4 and membranes (0.1 to 0.2 mg of protein per ml). ApH generation was initiated by addition of ascorbate (20 mM) and TMPD (100 to 300 FuM). Monensin and nonactin were used at the final concentrations given above. Analytical procedures. Protein concentrations were estimated by the method of Lowry et al. (25) with bovine serum albumin as a standard. The concentration of Na+ in the various buffers was determined with a Varian-Technikon AA120 atomic absorption spectrophotometer calibrated with different Na+ concentrations in distilled water. Materials. The following uniformly "'C-labeled amino acids were used: L-alanine (5.7 GBq/mol), L-glutamate (10.5 GBq/mol), L-leucine (11.8 GBq/mol), and L-lysine (11.7 GBq/mol). All radioactive chemicals were obtained from the

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Timeo (min) FIG. 3. Effect of ionophores on the uptake Of L-glutamate in membrane vesicles of B. stearothermophilus. Uptake of L-glutamate (1.75 ,M) was measured as described in the legend to Fig. 1. The figure shows L-glutamate uptake in the absence of electron donors (0), in the presence of the electron donors ascorbate (20 mM) and TMPD (300 puM) without ionophores (0), and in the presence of monensin (20 nM) (U) or nonactin (10 ,uM) (O). Final protein concentration, 0.28 mg/ml.

Radiochemical Centre, Amersham, United Kingdom. Pyranine was from Eastman Kodak Co. (Rochester, N.Y.), and diSC(3)5 was from Molecular Probes, Inc. (Junction City, Oreg.). RESULTS

Generation of a proton motive force in membrane vesicles. Transport of various amino acids in membrane vesicles of B. stearothermophilus was studied. Since the proton motive force was found to be a major driving force for amino acid transport, the influence of the medium composition on the Ap was studied. In previous publications de Vrij et al. (9, 10) demonstrated that in potassium phosphate (pH 6.0), oxidation via the respiratory chain of the electron donor ascorbate-TMPD results, in B. stearothermophilus membrane vesicles, in the generation of a high membrane potential (Aj = -105 mV) and a pH gradient (-ZA&pH = -45 mV). Qualitative information about the membrane potential generated has been obtained with the indicator diSC(3)5 (data not shown). Upon addition of the electron donor system ascorbate-TMPD to membrane vesicles suspended in 50 mM MES-NaOH (pH 6.0), a transient generation of the membrane potential was observed. The transient phase could be abolished by the addition of the Na+/H+ exchanger monensin (20 nM) and resulted in a slightly higher steady-state membrane potential. This transient phase is most likely due

794

HEYNE ET AL.

J. BACTERIOL.

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Time (sec) Time (sec) FIG. 4. Uptake of L-alanine in membrane vesicles of B. stearothermophilus driven by artificial gradients. Gradients were imposed at an external pH of 6.0 at 45°C as described in Materials and Methods, with a final concentration of 2.87 FM L-['4C]alanine. (A) Uptake of L-alanine driven by an artificial Ap (A), A/RNa+IF (0), Ap and APNa+/F (O), or ApH (A). Control experiments were performed by 100-fold dilution of membrane vesicles into the same buffer as that in which they were suspended (0). (B) Uptake of L-alanine driven by an artificial A, (U), At and ApRNa+/F (0), or ApH and APRNa+/F (C1). Control experiments were performed as described for panel A (0).

an interconversion of membrane potential into a pH gradient. The addition of either the Na+ ionophore nonactin (10 ,uM) or the protonophore SF-6847 (1 ,uM) resulted in a complete collapse of the membrane potential. In membrane vesicles in 50 mM MES-KOH (pH 6.0), ascorbate-TMPD oxidation also resulted in the generation of a transient membrane potential, but a significantly lower steady-state A4i was reached (data not shown). Addition of the K+/H+ exchanger nigericin (20 nM) increased the membrane potential drastically. To discriminate between a A4-depolarizing effect of K+ and a A4,-increasing effect of Na+ via Na+/H+ exchange activity, the effects of different monovalent cations on A&i generation in membrane vesicles in 50 mM Tris hydrochloride (pH 7.0) (contaminating [Na+], approximately 5 to 6 ,uM) were studied. The addition of KCI (50 mM) led to a significant decrease of the A+p, while the monovalent cations Na+, Li', Cs', Rb+, and NH4' did not significantly alter the membrane potential (data not shown). These observations indicate that K+ has a Adj-depolarizing effect which could be due to electrogenic K+ uptake. Effect of ionophores on amino acid uptake in membrane vesicles. The mechanisms of amino acid transport in B. stearothermophilus membrane vesicles were studied by investigating the role of the Ap, membrane potential, pH gradient, or sodium gradient in the uptake processes under defined conditions (i.e., a controlled sodium ion concentration) in 50 mM MES-NaOH (pH 6.0; supplemented with 10 mM MgSO4) at 45°C. In the presence of ascorbate-TMPD, Lalanine was accumulated by the membrane vesicles at a high rate to a high accumulation level (Ap4Ala/F = 93 mV) (Fig. 1A). Nonactin (10 ,uM) strongly decreased (APuAla/F < 30 mV) the uptake of alanine. Nonactin dissipated both the membrane potential and the Na+ gradient and increased the

to

ApH (as could be concluded from the fluorescence changes of pyranine [data not shown]). These observations indicate that alanine is most likely not symported with protons. The cotranslocated ion could be identified by studying the effect of the Na+/H+ exchanger monensin on the uptake of alanine. Monensin interconverts the pH gradient into a ARuNa+, and subsequent proton pumping results in an increase of the membrane potential (data not shown). Addition of monensin (20 nM) enhanced the initial uptake rate and the steady-state accumulation level (AIXAla/F > 112 mV) (Fig. 1A). These observations indicate that alanine is translocated in symport with Na+. Ascorbate-TMPD oxidation by membrane vesicles could also drive the uptake of L-leucine (Fig. 1B). Transport of this amino acid showed the same response to additions of monensin (20 nM) and nonactin (10 ,uM) as did L-alanine transport. Leucine uptake was not observed in the presence of a ApH alone. These observations indicate that also leucine is transported by an Na+-leucine symport mechanism. Transport of L-lysine was also studied. L-Lysine was transported by the membrane vesicles when a Ap was generated by ascorbate-TMPD oxidation (Fig. 2). The rate of L-lysine uptake was rather low. A steady-state level of lysine accumulation was not reached even after 11 min (AlILySIF > 133 mV). In this case, monensin (20 nM) did not stimulate the initial rate of uptake or steady-state accumulation of lysine, while the sodium ionophore nonactin (10 puM) or the protonophore SF-6847 (100 nM) completely inhibited the uptake of lysine. Since no uptake of lysine was found in the presence of a ApH alone, transport of L-lysine does not appear to occur in symport with H+ or Na+ but is most likely mediated by an uniport system. Upon the addition of ascorbate-TMPD, membrane vesi-

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Time (sec) Time (sec) FIG. 5. Uptake of L-leucine in membrane vesicles of B. stearothermophilus driven by artificial gradients. Gradients were imposed at an external pH of 6.0 at 45°C as described in Materials and Methods, with a final concentration of 1.49 p.M L-['4C]leucine. (A) Uptake of L-leucine driven by an artificial Ap (A), Ap'Na+/F (0), Ap and ApRNa+IF (O), or ApH (A). Control experiments were performed by 100-fold dilution of membrane vesicles into the same buffer as that in which they were suspended (0). (B) Uptake of L-leucine driven by an artificial A4i (U), A% and ApUNa+IF (0), or ApH and APRNa+IF (LI). Control experiments were measured as described for panel A (0).

cles also rapidly accumulated L-glutamate to a high level (A.LGlU/F = 160 mV) (Fig. 3). Previous studies have shown that L-glutamate is transported in symport with one H+ ion and one Na+ ion (9). Monensin (20 nM) enhanced both the initial uptake rate and the steady-state accumulation (AL.GlU/F > 190 mV) (Fig. 3). A decrease of the initial uptake rate and steady-state accumulation (AILGIU/F = 110 mV) of L-glutamate was observed in the presence of the sodium ionophore nonactin. The protonophore SF-6847 (100 nM) completely abolished the uptake of L-glutamate (Fig. 3). These results are consistent with an Na'-H+-L-glutamate symport system. The results also indicate that ascorbateTMPD oxidation is not coupled to Na+ translocation (see also reference 9). L-Alanine and L-leucine uptake driven by artificial gradients. Since studies with ionophores on amino acid uptake supply only indirect information about the mechanism of transport, the uptake of alanine and leucine was further analyzed under conditions in which a Aip, a ApH, an Na+ gradient, or a combination of these driving forces was artificially imposed. Alanine (Fig. 4) and leucine (Fig. 5) were transported only when a Alt, an Na+ gradient, or both were imposed. Uptake of alanine or leucine occurred at a higher rate in the presence of an Na+ gradient than in the presence of a A+. When both an Na+ gradient and a A/p were applied, uptake occurred at even more enhanced rates (Fig. 4A and SA). Combinations of A4i or Ap.Na+IF with ApH did not lead to enhanced rates of uptake. Alanine (Fig. 4B) and leucine (Fig. SB) were not taken up in the presence of a pH gradient alone, even when high concentrations of Na+ were present (20 mM; [Na+]in = [Na+]ou, [data not shown]). These observations indicate that the uptake of the neutral

amino acids alanine and leucine is an electrogenic process and that transport occurs in symport with a Na+ ion. Kinetic constants of the amino acid carriers. The kinetic constants and substrate specificities of the amino acid carriers were determined at 50°C in 50 mM Tris hydrochloride (pH 7.0) supplemented with 50 mM NaCl (Table 1). The membrane vesicles exhibit a high-affinity transport system for L-glutamate (K, = 4.7 ,uM) with a high maximal velocity (Vmax = 11.4 nmol/min/mg of protein). L-Glutamate transport was competitively inhibited by L-aspartate (K, 3.8 p.M), but a 30-fold excess of the amino acids L-glutamine, L-asparagine, and D-glutamate did not inhibit L-glutamate transport significantly (data not shown). The transport system for L-leucine has a high affinity for this amino acid (Kt = 0.9 ,uM [Table 1]). Transport of leucine was competitively inhibited by L-valine (Ki = 2.5 ,uM) and L-isoleucine (Ki = 1.0 ,uM). The transport system for L-alanine has a high affinity for this amino acid (K, = 12.1 ,uM). L-Serine appeared to be the most effective inhibitor (Kl = 49.0 jiM), and only slight inhibition of L-alanine transport was observed in the presence of a 10- to 30-fold excess of L-glycine (Ki > 300 FM) and L-threonine (Ki = 160.0 ,uM). L-Lysine is transported via a relatively low-affinity (K, = 12.9 ,uM [Table 1]) stereospecific transport system with a low maximal velocity (Vmax = 0.5 nmol/min/mg of protein). L-Lysine transport was competitively inhibited by L-arginine (Ki = 3.4 F.M [Table 1]) but not by a 20-fold excess of L-ornithine, D-lysine, or the substrate analogs 8-hydroxylysine, D,L-2,3-diamino-propionic acid, L-2,4-diamino-n-butyric acid, and S-2-aminoethyl-L-cysteine (data not shown). Cation dependency and specificity of the amino acid transport systems. The effects of different monovalent cations on =

7%

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HEYNE ET AL.

TABLE 1. Kinetic constants for transport of amino acids by membrane vesicles of B. stearothermophilusa Transported substrate

L-Leucine L-Alanine

L-Lysine L-Glutamate

Ki (LM) of inhibitor'

K,

Vmax (nmol/min/mg of

(>M)

protein)

lle

Val

Ser

Thr

Gly

Arg

Asp

0.9 12.1 12.9 4.7

1.2 7.2 0.5 11.4

1.0 ND ND ND

2.5 ND ND ND

ND 49.0 ND ND

ND 160.0 ND ND

ND >300 ND ND

ND ND 3.4 ND

ND ND ND 3.8

a Kinetic parameters were extrapolated from the initial rate of uptake, measured at different substrate concentrations, by Eadie-Hofstee analysis. Uptake was measured at 50°C in 50 mM Tris hydrochloride (pH 7.0) supplemented with 50 mM NaCl and 10 mM MgSO4. b Inhibitor constants were estimated from the inhibition of amino acid uptake (at concentrations below the K,) with a 10- to 30-fold excess of inhibitor by the equation given in Materials and Methods. The K, was averaged from three determinations. ND, Not determined.

the initial uptake rates of the various amino acids in a buffer with a low concentration of sodium ions were studied. The sodium contamination in the various buffers was determined by atomic absorption spectroscopy. Minimal contamination with Na+ was found in 50 mM Tris hydrochloride (pH 7.0-10 mM MgSO4. Very low rates of alanine or leucine uptake in this buffer were observed after the addition of ascorbate-TMPD (Fig. 6). The rates of uptake of alanine (Fig. 6A) and leucine (Fig. 6B) were strongly stimulated when the buffer was supplemented with 50 mM NaCl. The addition of 50 mM chloride salts of the monovalent cation K+, Rb+, Cs', or NH4' did not stimulate the uptake rate of alanine or leucine. Only Li' stimulated the uptake rate to some extent. 0.40

To examine the Na+ dependency in more detail, the effect of different concentrations of Na+ on the initial rates of uptake of alanine, leucine, and glutamate was studied. The initial rates of uptake for alanine (Fig. 7A) and leucine (Fig. 7B) display Michaelis-Menten kinetics with respect to the sodium ion concentration. However, high concentrations of sodium ions ([Na+] = 50 mM) reduce the initial uptake rate. Data (up to 10 mM Na+) were analyzed by Eadie-Hofstee transformation (Fig. 7, insets) and revealed a low affinity of the alanine carrier for Na+ (K, = 1.0 mM), whereas the leucine carrier has a somewhat higher affinity for Na+ (K, = 0.4 mM). In contrast, a high initial uptake rate of L-glutamate was found when no Na+ was added (data not shown). Increasing concentrations of sodium ions only slightly stim60

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time (sec) time (8ec) FIG. 6. Cation dependency of L-alanine and L-leucine transport. (A) Uptake of L-alanine (2.87 ,uM) was measured at 50°C in membrane vesicles of B. stearothermophilus (0.34 mg of protein per ml) which were washed and resuspended in 50 mM Tris hydrochloride (pH 7.0)-10 mM MgSO4 containing the electron donor ascorbate-Tris (20 mM) and TMPD (100 ,uM) in the presence of NaCl (0), KCI (0), LiCl (U), or CsCl (O). The cations were added up to a final concentration of 50 mM. The sodium ion concentration present in 50 mM Tris hydrochloride (pH 7.0) as a contaminant was 5 to 6 ,UM. (B) Uptake of L-leucine (1.57 ,M) in membrane vesicles of B. stearothermophilus (0.49 mg of protein per ml) was measured under the conditions described for panel A.

VOL. 173, 1991

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ulated the initial uptake rate of L-glutamate (data not shown), indicating an extremely high affinity of the glutamate carrier for sodium ions (K, =

0.20

0.00

=

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Despite the absence of participation of an

Na' 0.32

H+

ion in the

translocation process of alanine, the transport activity driven by an artificial membrane potential and sodium gra-

concentration. (mM)

dient showed a pH dependency remarkably similar to that of

B

the glutamate transport activity (Fig. 8B). The uptake rate of alanine decreased with increasing pH (pKa = 7.6). DISCUSSION

0.24 co

E

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;

In this report we have shown that neutral amino acids are transported by B. stearothermophilus membrane vesicles via an Na+ symport system. Several experimental data support this conclusion. Uptake of the neutral amino acids (L-alanine

and L-leucine) is driven by one of the components of the Ap, but an H+ symport mechanism for these amino acids is not .C likely because a collapse of the Na+ gradient by nonactin 0.16 2 without a collapse of the Ap leads to a complete inhibition of / ,jg 0.20 . \ transport. On the other hand, an interconversion of ApH into E A\\Na+ and an increase of the membrane potential by the s \ addition of monensin have a stimulatory effect on the steadyc state accumulation levels of both the neutral and acidic acids. Furthermore, only an artificially imposed amino a > ( | [ 0.10 \ 0 *A\LNa+, or both act as a driving force for the uptake of \ 0.08 L-alanine and L-leucine. These observations indicate that the neutral amino acids are symported with Na+ ions rather than with protons. The low uptake rate of these amino acids in the _j o.oo presence of only an artificial Atp can be explained by the low 0.00 0.20 0.40 0.60 0.80 1.00 contamination of sodium ions (approximately 21 to 22 FiM) in V/s (nmol ieucnm/minmwmgu) the uptake medium (choline-MES). The initial uptake rates A . 0.00 . * of L-alanine and L-leucine increase with increasing [Na+] .' * t 0 3 6 9 50

_.08

Na+ concentration (mM) FIG. 7. Sodium ion dependency of L-alanine (A) and L-leucine (B) transport. The initial rates of L-alanine uptake (2.87 pLM) and L-leucine uptake (1.57 ,uM) in B. stearothermophilus membrane vesicles (0.68 mg of protein per ml; washed and resuspended in 50

A/v,

mM Tris hydrochloride (pH 7.0)-10 mM MgSO4) were determined from the initial rate of uptake at 50°C. Uptake was measured in the presence of different concentrations of NaCl (0 to 50 mM). (Insets) Eadie-Hofstee plots of the data.

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J. BACTERIOL.

0.50

0.35

A

B

0

0.30

0.40 . 40

0

a

~1

0

0.25 0

0.30 .

0

la

'a

q

0.20

I 0.20 .

hi

0i

0.15

0.10 . 0.10

0.00

.

5

*-

X

6

7

a

I

a

9

0.05 10

*

5

*

6

*

* * aX

7

*

a

9

10

external pH oxtornal pH FIG. 8. Dependency of the initial rate of L-glutamate or L-alanine uptake in membrane vesicles of B. stearothermophilus on the external pH. The initial rates of L-glutamate uptake (1.75 ,uM [A]) and L-alanine uptake (2.87 ,uM [B]) were determined at various external pHs (5.5 to 9.0) by imposing (at 45°C) an artificial A and Ap,Na/+F as described in Materials and Methods.

according to Michaelis-Menten saturation kinetics, which is also in accordance with an Na+ symport mechanism. Competition experiments with membrane vesicles of B. stearothermophilus indicate that the acidic amino acids L-glutamate and L-aspartate are transported via a specific transport system. Transport of these amino acids occurs in a 1:1:1 stoichiometry with H+ and Na+, in analogy to what has been observed in E. coli B (13, 14). The Michaelis constant of glutamate transport in E. coli B has been shown to depend strongly on the concentrations of both Na+ and H+. The rate of L-glutamate transport in B. stearothermophilus decreases TABLE 2. Effect of external pH on kinetic parameters of the glutamate transport system in B. stearothermophilusa External pH

K, (,uM)

Vmax (nmol/min/mg of protein)

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

2.6 5.7 3.4 1.8 16.6 10.0 21.1 38.9 5.4

0.11 1.29 2.0 1.33 0.48 2.15 1.53 2.2 0.47

a A concentrated vesicle suspension in 100 mM potassium acetate-20 mM MES-20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-20 mM Tris of appropriate pH was diluted 100-fold into 100 mM sodium acetate-20 mM MES-20 mM HEPES-20 mM Tris of the same pH in the presence of various concentrations (0.72 to 21.75 ,uM) of L-[14C]glutamate at 50°C. The kinetic parameters were extrapolated from the initial rate of uptake by Eadie-Hofstee analysis and were averaged from three determinations.

with increasing pH (decrease of proton concentration) (PKa = 6.9). The relative concentrations of the different species of glutamate between pH 4.0 and 7.0 are mainly determined by the protonation state of the y-carboxyl group. The concentrations of glutamic acid and the glutamate anion can be calculated at a given pH with the Henderson-Hasselbach equation. The apparent affinity constant (K,) of the transport system should increase exponentially with increasing pH when glutamic acid is the only transported substrate of the glutamate transport system. Such a pH effect on the K, of glutamate transport has been found for Lactococcus lactis (27). The pH dependence of the K, for L-glutamate transport in B. stearothermophilus indicates that the glutamate anion is possibly the transported substrate. Experiments were performed to discriminate between the effects of the external pH and internal pH on the uptake of L-glutamate. At a fixed external pH, the ApH is always a less effective driving force of L-glutamate uptake than the Ai is (data not shown). When the glutamate anion is transported with one Na+ ion and one H+ ion, one would expect both driving forces to be equally effective. However, in the presence of a ApH, the internal pH is higher than the external pH, and this increase in internal pH can result in a decrease in the rate of L-glutamate uptake. Such internal pH-dependent transport systems have also been described for Lactococcus lactis subsp. lactis (27), L. lactis subsp. cremoris (12), and Rhodobacter sphaeroides (1). Since the rate of deprotonation of the carrier on the inner surface of the membrane would increase with increasing pH and thus would increase the rate of H+/Na+-linked L-glutamate uptake, the pHin most likely affects the transport

VOL. 173, 1991

BACILLUS Na+-DEPENDENT AMINO ACID TRANSPORT

system allosterically. This also appears to be the case with alanine transport. This transport activity decreased with increasing external pH (PKa = 7.6), while no protons participated in the translocation process of L-alanine. The L-alanine transport system in B. stearothermophilus is rather specific for L-alanine and for the cations Na+ and Li'. Only slight inhibition of L-alanine transport is observed with L-serine. From the related thermophile PS3, an L-alanine transport system has been purified and functionally reconstituted into liposomes (18-22). An apparent K, of 14 ,uM for L-alanine transport was found; this value is close to the value of 12.1 puM obtained for B. stearothermophilus. However, in contrast to what has been observed for L-alanine transport in membrane vesicles from B. stearothermophilus, transport of L-alanine in PS3 is competitively inhibited by L-glycine (18). Na+-dependent L-leucine transport has also been observed in B. stearothermophilus membrane vesicles. Transport of L-leucine is competitively inhibited by L-isoleucine and L-valine, suggesting that both L-isoleucine and L-valine are substrates of the leucine carrier. Similar cation and solute specificities have been observed in Pseudomonas aeruginosa (34) and the facultatively alkalophilic Bacillus strain YN-2000 (35). In the latter organism, transport of the neutral branched amino acids is facilitated by an Na+ mechanism over a broad pH range (pH 7.0 to 10.0). The Na+/H+ exchanger monensin has no significant stimulating effect on the uptake rate and steady-state accumulation level of L-lysine. If lysine were transported with one Na+ ion, the driving force for uptake would be equivalent to 2A4* + AP,Na+/F. In the presence of monensin, the thermodynamic equilibrium should then exceed -200 mV, which has not been observed (Fig. 5). L-Lysine uptake was also not stimulated by the cations Na+ and Li+. These results indicate that the transport system of the cationic amino acids L-lysine and L-arginine is not Na+ dependent and is most likely a uniport system. The L-glutamate/L-aspartate transport system possesses a high affinity for Na+ (K, < 5 ,uM), while L-alanine (K, = 1.0 mM) and L-leucine (K, = 0.4 mM) systems have rather low affinities. One could speculate about whether the neutral amino acid carriers possess regulatory as well as catalytic binding sites for Na+, as has been suggested by Russell et al. (28) for Na+-dependent neutral amino acid transport in S. bovis. Hill plots for the L-alanine (napp = 1.52 + 0.57) and the L-leucine (napp = 1.43 + 0.38) transport systems do not indicate that the neutral transport systems have one or more binding sites for Na+. High Na+ concentrations ([Na+] = 50 mM) inhibited both L-alanine and L-leucine transport. Inhibition by high Na+ concentrations has also been observed for the Na+-coupled L-proline and L-serine/L-threonine transport system in E. coli (6, 16). Potassium depolarizes the membrane potential in membrane vesicles from B. stearothermophilus (data not shown). This depolarization can be caused by electrogenic K+ transport, as has been found in a variety of bacteria, by K+/H+ antiporters, or by specific K+ transport systems (1, 2, 4). An alternative explanation can be given for the observed phenomenon. It has been observed that potassium inhibits Na+/H+ exchange activity competitively in Methanobacterium thermoautotrophicum (29). Such an inhibition in B. stearothermophilus would facilitate the generation of a significant ApH and lead to a lower A+. The specific lowering of the A* by potassium in membrane vesicles from B. stearothermophilus can be partially prevented by the addition of nigericin, an ionophore which specifically collapses the

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ApH. This indicates that the membrane potential and the pH gradient across the membrane can to some extent be interconverted. From the data described above, it is difficult to conclude that Na+/H' exchange activity exists in the membrane vesicles. Goto et al. (15) noted Na+/H+ exchange activity in the related thermophile PS3. Such activity would supply the chemical gradient of sodium ions by interconversion of the ApH into a sodium gradient. ACKNOWLEDGMENT We thank D. Molenaar for valuable discussions. REFERENCES 1. Abee, T., K. J. Hellingwerf, and W. N. Konings. 1988. Effects of potassium ions on proton motive force in Rhodobacter sphaeroides. J. Bacteriol. 170:5647-5653. 2. Bakker, E. P., I. R. Booth, U. Dinnbier, W. Epstein, and A. Gajewska. 1987. Evidence for multiple K+ export systems in Escherichia coli. J. Bacteriol. 169:3743-3749. 3. Bassilana, M., T. Pourcher, and G. Leblanc. 1987. Facilitated diffusion properties of melibiose permease in E. coli membrane vesicles. J. Biol. Chem. 262:16865-16870. 4. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49:359-378. 5. Cairney, J., C. F. Higgins, and I. R. Booth. 1984. Proline transport through the major transport system of Salmonella typhimurium is coupled to sodium ions. J. Bacteriol. 160:22-27. 6. Chen, C. C., and T. H. Wilson. 1986. Solubilization and functiohnal reconstitution of the proline transport system of Escherichia coli. J. Biol. Chem. 259:7791-7796. 7. Clement, N. R., and M. J. Gould. 1981. Pyranine (8-hydroxy1,3,6-pyrenotrisulfate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles. Biochemistry 20:

1534-1538. 8. Damiano, E., M. Bassilana, J.-L. Rigaud, and G. Leblanc. 1984. Use of the pH sensitive fluorescence probe pyranine to monitor internal pH changes in Escherichia coli membrane vesicles. FEBS Lett. 166:120-124. 9. de Vrij, W., R. A. Buithuis, P. R. van Iwaarden, and W. N. Konings. 1989. Mechanism of L-glutamate transport in membrane vesicles from Bacillus stearothermophilus. J. Bacteriol. 171:1118-1125. 10. de Vrij, W., R. I. R. Heyne, and W. N. Konings. 1989. Characterization and application of a thermophilic primary transport system: cytochrome-c oxidase from B. stearothermophilus. Eur. J. Biochem. 178:763-770. 11. Dimroth, P. 1987. Sodium ion transport decarboxylases and other aspects of sodium ion cycling in bacteria. Microbiol. Rev.

51:320-340. 12. Driessen, A. J. M., J. Kodde, S. de Jong, and W. N. Konings. 1987. Neutral amino acid transport by membrane vesicles of Streptococcus cremoris is subjected to regulation by internal pH. J. Bacteriol. 169:2748-2754. 13. Fujimura, T., I. Yamato, and Y. Anraku. 1983. Mechanism of glutamate transport in E. coli B. 1. Proton-dependent and sodium-ion dependent binding of glutamate to a glutamate carrier in the cytoplasmic membrane. Biochemistry 22:19541959. 14. Fujimura, T., I. Yamato, and Y. Anraku. 1983. Mechanism of glutamate transport in E. coli B. 2. Kinetics of glutamate transport driven by artificially imposed proton and sodium ion gradients across the cytoplasmic membrane. Biochemistry 22: 1959-1965. 15. Goto, K., H. Hirata, and Y. Kagawa. 1980. A stable Na+/H+ antiporter of thermophilic bacterium PS3. J. Bioenerg. Biomembr. 12:297-308. 16. Hama, H., T. Shimamoto, M. Tsuda, and T. Tsuchiya. 1987. Properties of Na+-coupled serine-threonine transport system in Escherichia coli. Biochim. Biophys. Acta 905:231-239. 17. Heefner, D. L., and F. M. Harold. 1982. ATP-driven sodium pump in Streptococcus faecalis. Proc. Natl. Acad. Sci. USA

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79:2798-2802. 18. Hirata, H. 1979. Characterization of alanine transport by reconstituted proteoliposomes, p. 505-512. In E. Quagliariello et al. (ed.), Function and molecular aspects of biomembrane transport. Elsevier/North-Holland Biomedical Press, Amsterdam. 19. Hirata, H., T. Kambe, and Y. Kagawa. 1984. A purified alanine carrier composed of a single polypeptide from thermophilic bacterium PS3 driven by either proton or sodium ion gradient. J. Biol. Chem. 259:10653-10656. 20. Hirata, H., N. Sone, H. Yoshida, and Y. Kagawa. 1976. Solubilization and partial purification of alanine carrier from membranes of a thermophilic bacterium and its reconstitution into functional vesicles. Biochem. Biophys. Res. Commun. 69:665671. 21. Hirata, H., N. Sone, H. Yoshida, and Y. Kagawa. 1976. Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium. J. Biochem. 79:1157-1166. 22. Hirata, H., N. Sone, H. Yoshida, and Y. Kagawa. 1977. Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport. J. Supramol. Struct. 6:77-84. 23. Kitada, M., A. A. Guffanti, and T. A. Krulwich. 1982. Bioenergetic properties and viability of alkalophilic Bacillusfirmus RAB as a function of pH and Na+ contents of the incubation medium. J. Bacteriol. 152:1096-1104. 24. Konings, W. N., A. Bisschop, M. Veenhuis, and C. A. Vermeulen. 1973. New procedure for the isolation of membrane vesicles of Bacillus subtilis and an electron microscopy study of their ultrastructure. J. Bacteriol. 116:1456-1465. 25. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 26. Padan, E., D. Zilberstein, and S. Schuldiner. 1981. pH homeo-

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stasis in bacteria. Biochim. Biophys. Acta 650:151-166. 27. Poolman, B., K. J. Hellingwerf, and W. N. Konings. 1987. Regulation of the glutamate-glutamine transport system by intracellular pH in Streptococcus lactis. J. Bacteriol. 169:22722276. 28. Russell, J. B., H. J. Strobel, A. J. M. Driessen, and W. N. Konings. 1988. Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium. J. Bacteriol. 170:3531-3536. 29. Schonheit, P., and D. B. Beimborn. 1985. Presence of Na+/H+ antiporter in Methanobacterium thermoautotrophicum and its role in Na+ dependent methanogenesis. Arch. Microbiol. 142: 354-361. 30. Skulachev, V. P. 1985. Membrane-linked energy transductions. Bioenergetic functions of sodium: H' is not unique as a coupling ion. Eur. J. Biochem. 151:199-208. 31. Speelmans, G., W. de Vrij, and W. N. Konings. 1989. Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus. J. Bacteriol. 171:3788-3795. 32. Tokuda, H., and T. Unemoto. 1981. A respiratory-dependent primary extrusion system functioning at alkaline pH in the marine bacterium Vibrio alginolyticus. Biochem. Biophys. Res. Commun. 102:265-271. 33. Tsuchiya, T., and T. H. Wilson. 1978. Cation-sugar cotransport in the melibiose transport system of Escherichia coli. Membr. Biochem. 2:63-77. 34. Uratani, Y. 1985. Solubilization and reconstitution of sodiumdependent transport for branched-chain amino acids from Pseudomonas aeruginosa. J. Biol. Chem. 260:10023-10026. 35. Wakabayashi, K., N. Koyama, and Y. Nosoh. 1988. Leucine transport system in a facultatively alkalophilic Bacillus. Arch. Biochem. Biophys. 262:19-26.