Vol. 123, No. 1
JOURNAL OF BACTERoLOGY, July 1975, p. 123-127 Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Active Transport of Manganese in Isolated Membrane Vesicles of Bacillus subtilis PINAKILAL BHATFACHARYYA' Department of Biology, Washington University, St. Louis, Missouri 63130 Received for publication 14 April 1975
Membrane vesicles isolated from cells of Bacillus subtilis W23 accumulate in the presence of an energy source. The artificial electron donor system ascorbate and phenazine methosulfate or reduced nicotinamide adenine dinucleotide and phenazine methosulfate can supply the energy for the uptake. D-Lactate in the presence or absence of phenazine methosulfate would not support manganese accumulation. Anaerobiosis, cyanide, m-chlorophenyl carbonylcyanide hydrazone, valinomycin, gramicidin, and p-hydroxy-mercuribenzoate inhibit the uptake. The inhibition by p-hydroxymercuribenzoate is prevented by excess dithiothreitol. Potassium fluoride or sodium arsenate has no effect on the uptake. The manganese transport system in the B. subtilis vesicles exhibits Michaelis-Menten kinetics with a Km of 13 gM and a Vmax of 1.7 nmol/min per mg (dry weight) of membranes. The uptake of manganese is specific and is not inhibited by 0.1 mM CaCl2 or MgCl2. manganese
Studies with isolated membrane vesicles have revealed that active transport systems for a number of structurally different compounds including sugars (1, 5, 9, 11), amino acids (4, 12, 13, 15, 18), manganese (2), and potassium (3, 16) reside in the cytoplasmic membranes of different organisms. The transport systems may be coupled to either the phosphoenolpyruvate phosphotransferase system (in the case of glucose and fructose) or to a membrane-bound dehydrogenase (1, 9, 11, 12, 15). The energy substrate for dehydrogenase-coupled transport differs in the membrane vesicles from different organisms depending upon the nature of the organism and conditions of growth. Where Dlactate is the most effective energy source for transport in Escherichia coli and Micrococcus denitrificans membranes (1, 11, 12), transport of amino acids in Staphylococcus aureus membranes is coupled to a-glycerophosphate dehydrogenase (18) and in Bacillus subtilis membranes to reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase or a-glycerophosphate dehydrogenase, if the cells are grown in glycerol (15). The present paper describes an energy-dependent manganese accumulation system in the membrane vesicles from log-phase cells of B. subtilis W23 in the presence of NADH-phenazine methosulfate (PMS) or as' Present address: Marrs McLean Department of Biochemistry, Baylor College of Medicine, Texas Medical Center, Houston, Tex. 77025.
123
corbate-PMS. The corresponding transport system in the whole cells of B. subtilis has been recently reported from this laboratory (6, 7). MATERIALS AND METHODS Organism and media. B. subtilis strain W23 was used. Cells were maintained on tryptone agar and grown in tryptone broth (6) or in tryptone broth supplemented with 25 mM KCl and CaCl2 and MgCl2, each 1.0 mM. Preparation of membrane vesicles. In early experiments, vesicles were prepared by the method of Kaback (10) as modified and described by Konings and Freese (15). Later, the membrane vesicles were prepared by a modified and simpler method as developed and communicated by Konings et al. (14). Cells were harvested from mid-log phase of growth and washed twice with 0.1 M K-PO4 buffer (pH 7.3). It is important to harvest the cells before they reach the stationary phase of growth since stationary phase cells give inactive or defective vesicles. The pellet was then resuspended thoroughly in 50 mM K-PO4 (pH 8.0) buffer and diluted into a large volume of 50 mM K-PO4 buffer (pH 8.0) containing 300 Mg of lysozyme per ml, 10 Asg of deoxyribonuclease per ml, 10 Ag of ribonuclease per ml, and 10 mM MgSO4. The volume of this incubation mixture was approximately 300 mVg (wet weight) of cells or at least one-third of the original growth medium volume. The diluted cells were then incubated for 45 min at 37 C. Ethylenediaminetetraacetate (K-salt, adjusted to pH 8.0 with KOH) was then added to 15 mM final concentration, and the mixture was further incubated for 15 min, followed by an additional 30-min incubation after the addition of 20 mM MgSO4. Cells, by now converted
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into vesicles, were then centrifuged at 16,000 x g for 30 to 45 min at 4 C. The pellet was washed twice with 0.1 M K-PO4 buffer (pH 6.6) with repeated homogenization and centrifugation. If the pellet was sticky, it was suspended in 50 ml of 0.1 M K-PO4 buffer (pH 6.6) containing 10 ,gg each of deoxyribonuclease and ribonuclease per ml and 10 mM MgSO4 and incubated for 30 min with stirring. Before collecting the final pellet at 34,000 x g for 30 min, two low-speed contrifugation pellets (800 x g) were discarded. The 34,000 x g pellet was then suspended in 0.25 M sucrose by homogenization and again centrifuged. The vesicles were then resuspended in 0.25 M sucrose to an approximate Klett reading of 700 (ca. 540 nm, green filter), frozen in 1.0- or 0.5-ml portions in a dry ice-acetone bath, and stored at -80 C. Manganese uptake by B. subtilis vesicles. Frozen vesicles were thawed at 30 C and diluted with 0.25 M sucrose. Appropriate amounts were then distributed into several disposable culture tubes (10 by 75 mm), and additions of different substances were made so that the final volume of the mixture was 0.1 ml and the composition was as described in the figure legends. During incubation, the tubes were placed in a water bath over a magnetic stirrer and stirred with Teflon-coated 7-mm magnets while either 0, or Nwas blown over the surface of the incubation mixture. At indicated times the tubes were taken out of the water bath, and the contents of the tubes were diluted with 2 ml of 0.25 M LiCl and filtered through membrane filters (type HA; Millipore Corp., Bedford, Mass.) and washed further with 2 ml of 0.25 M LiCl. Washing with LiCl gives optimum results with transport studies in membrane vesicles (1), and 0.25 M LiCl gives optimum results for overall manganese uptake under the experimental conditions. The radioactivity on the filters was then determined in a scintillation spectrometer as described earlier (19). Each point was corrected for a background obtained by diluting the contents of a control tube with LiCl before the addition of the radioactive manganese chloride. Uptake is expressed as nanomoles of manganese taken up per milligram (dry weight) of membranes. Materials. Reagent grade chemicals and deionized water were used in all experiments. D-(- )-Lactic acid (lithium salt), NADH (disodium salt), p-chloromercuribenzoate (sodium salt), and PMS were purchased from Sigma Chemical Co., St. Louis, Mo.; m-chlorophenyl carbonylcyanide hydrazone (CCCP) and valinomycin from Calbiochem, Los Angeles, Calif.; dithiothreitol from P. L. Biochemicals, Inc., Milwaukee, Wis.; and gramicidin from Mann Research Laboratory, New York, N.Y. Radioactive [4C Jproline and carrier-free radioactive 54MnCl, were purchased from New England Nuclear Corp., Boston, Mass.
PMS and oxygen were blown over the surface of the mixture. The addition of radioactive 54MnCl, led to rapid accumulation of the radioactivity inside the vesicles that reached a maximum at 10 min (Fig. 1). On further incubation, the accumulated manganese was released from the vesicles presumably after the exhaustion of the energy source (15). The maximal level of uptake at about 10 min does not represent cessation of manganese uptake altogether but an equilibrium of influx and efflux. Thus when the membranes were incubated with nonradioactive 10-6 M manganese chloride and a trace amount of radioactive 54MnCl, was added at 10 min, there was further uptake of radioactivity but with a lower rate and lower equilibrium level than when 54MnCl, was added at the beginning. Effects of energy substrates and metabolic inhibitors. Under anaerobic conditions, there was very little uptake of manganese by the B. subtilis vesicles (Fig. 2A). Omission of either PMS or ascorbate resulted in manganese accumulation no higher than that observed under anaerobic conditions. NADH did not support manganese uptake to any significant extent in the absence of PMS (data not shown), but in the presence of PMS the rate of manganese uptake was almost the same with either NADH or ascorbate. There was no uptake with D-lactate in the presence (Fig. 2A) or absence of PMS
RESULTS Accumulation of manganese by membranes. When the vesicles were suspended in 0.2 M sucrose in the presence of an artificial electron donor system, sodium ascorbate and
FIG. 1. Manganese accumulation by Bacillus subtilis membranes. The incubation medium contained 0.2 M sucrose, 20 mM sodium ascorbate, 0.1 mM PMS, and approximately 2 mg of membranes per ml. Nonradioactive 10- M MnCl, was added at 0 min and trace 54MnCI, was added at 0 min (0) or 10 min (0).
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FIG. 2. Effect of energy substrates and metabolic inhibitors on the manganese uptake. (A) The incubation medium contained 0.2 M sucrose, 10 mM K-PO4 buffer (pH 6.6), 20 mM energy substrates, 0.1 mM PMS, and 3 mg of membranes per ml. "4MnCl, was added at 10- 6M. Symbols: 0, ascorbate + PMS + 0,; 0, ascorbate + PMS + N,; A, NADH + PMS + 0,, A, D-lactate + PMS + O;, 0, PMS + 0 - ascorbate; M, ascorbate + 0° - PMS. (B) The incubation mixture contained 0.2 M sucrose, 10 mM K-PO4 buffer (pH 6.6), 1 mM MgSO4, 20 mM sodium ascorbate, 0.1 mM PMS, and 3.3 mg of membranes per ml. Inhibitors were added immediately prior to the addition of "'MnCI,. Symbols: 0, control; 0, 10-6 M CCCP; A, 10-' M p-hydroxymercuribenzoate; A, 10-' M p-hydroxymercuribenzoate + 5 x 10-4 M dithiothreitol; 0, 10-2 M KCN; *, 2 x 10-6 M valinomycin; V, 2 x 10- 6 Mgramicidin.
(data not shown). Thus the energy for manganese accumulation by B. subtilis can be supplied by the oxidation of reduced PMS or NADH through the cytochrome chain. Figure 2B shows the effects of metabolic poisons on manganese uptake by the B. subtilis membranes. Potassium cyanide, an inhibitor of electron transport, inhibited manganese accumulation almost completely. A sulfhydryl reagent, p-hydroxymercuribenzoate, when added just prior to the addition of the radioactive 54MnCl2, inhibited the uptake about 40%, and the inhibition was antagonized by prior addition of 5 x 10-8 M dithiothreitol. Potassium fluoride (10-2 M) or sodium arsenate (1.6 x 10-2 M) did not affect the manganese uptake to any significant extent (data not shown). The proton conductor CCCP or ionophores like valinomycin and gramicidin inhibited manganese uptake very effectively (Fig. 2B). Kinetics of minganese transport. The initial rate of manganese uptake was determined in the presence of varying concentrations of
125
manganese (Fig. 3). Over the range from 1 to 24 ,M Mn2 , the manganese uptake in the vesicles showed Michaelis-Menten kinetics with a Km of 13 gM and Vmax of 1.7 nmol/min per mg of membranes. However, increasing the concentration of the substrate above 25 ,M, up to 200 ,M, never resulted in complete saturation of the system for manganese accumulation. Specificity of the manganese uptake. Near the micromolar range of manganese, the system is highly specific, and manganese uptake is not inhibited by magnesium or calcium (Fig. 4). When the incubation medium was made using either Na+ or K+ salts of ascorbate, addition of K+ or Na+ salts up to 10 mM concentration did not significantly alter the uptake of manganese (data not shown). However, ferrous chloride and cuprous chloride at 0.1 mM concentrations seriously inhibit the accumulation of radioactive manganese. The inhibition by these salts is likely due to inhibition of the energy metabolism in the membranes or to some nonspecific changes in the membranes, since proline uptake is also inhibited to a comparable extent (Fig. 4B). The values for proline uptake in the vesicles are considerably lower than those reported by Konings and Freese (15). Since the vesicles were prepared for the purpose of studying manganese uptake, they were washed and stored in sucrose. Proline uptake by such vesicles is poor.
/
1000
rE
~~~~~~~~0
0.004 ~~~~~0
1 * 0
5 0.003 a 0.02
0
j 0
8
16
24
[Manganesel [#M]
FIG. 3. Kinetics of manganese accumulation. Incubation medium contained 0.2 M sucrose, 10 mM tris(hydroxymethylaminomethane-hydrochloride (pH 7.0), 20 mM sodium ascorbate, 0.1 mM PMS, and 3 mg of membranes per ml. Temperature of incubation was 30 C, and the initial rates of uptake were determined over the first minute after the addition of
"4MnCI2.
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u2
0
+ ~ 0 +C
~
~
~
O
O
1
200
O~~~~~~~~~~~~~~~~~~O
2
3
0
2
4
minutes at 30 C
FIG. 4. Effect of cation on the uptake of manganese and proline. (A) Membranes were suspended at I mg/ml in 0.2 M sucrose, 20 mM sodium ascorbate, and 0. 1 mM PMS. 114MnCl, was added at 10- " M and nonradioactive salts at 10-4 M. Symbols: O,
control; *, CaCI,,, A, MgCI,, A, FeC1, O, CuCI, *,
MnCl.. (B) The incubation medium contained 0.2 M sucrose, 20 mM sodium ascorbate, 0.1 mM PMS, 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.0) and 1.3 mg of membranes per ml. Rad ioactive 1lm4Clproline was 2.4t added at M. DISCUSSION Manganese uptake in membrane vesicles of B. subtilis showed qualitatively similar properties to manganese active transport in intact cells (6), for example, requirement for energy metabolism, saturation kinetics, and substrate specificity. Similar observations were reported on manganese accumulation in membrane vesicles of E. coli (2). However, with respect to manganese transport, membrane vesicles of E. coli differ from those of B. subtilis in one significant aspect. Manganese uptake in E. coli membranes is dependent on the presence of CaClb in the incubation medium, whereas that in B. subtilis membranes is not. The kinetics studies of the manganese uptake in the B. subtilis membrane vesicles yielded a Kc of approximately 13 BM and Vsax of 17 nmol/min per mg of membranes. The corresponding values for the whole cells are 1 sM and 0.5 nmol/min per mg (6). Calculating on a rough basis, the Vax in the membrane vesicles is about one-third of that in the wholescells and compares favorably with similar determinations in other transport systems (2, 9, 11). The higher value for the KVmwith membrane vesicles does not mean that manganese transport by the membranes is significantly different from that
by the intact cells, since changes in cell wall shielding components would be expected to influence trace, low Vmax systems such as that for manganese especially strongly. Alternatively, components of the system may be altered during membrane preparation. Most of the accumulated manganese does not appear to be free, since adding trace 54MnCl2 after 10-min incubation with 10-6 M nonradioactive MnCl2 resulted in a lower equilibrium value for "4Mn uptake than that obtained when the radioactivity was added at the beginning of the incubation (Fig. 1). Furthermore, addition of excess nonradioactive manganese chloride or the uncoupling agent CCCP at any point during the active uptake of "4MnCl2 resulted in an immediate cessation of uptake of radioactivity but very little release of already accumulated radioactivity (data not shown). With levels of accumulation in the picomoles per milligram range, absorption to intramembrane polyanions could account for the lack of extensive exchange. The energy for the manganese uptake in the membrane vesicles from B. subtilis could be supplied by NADH and PMS or ascorbate and PMS but not by D-lactate. This confirms the observations of Konings and Freese (15) on energy sources for amino acid transport in B. subtilis membrane. The inhibition of the manganese uptake by anaerobiosis and KCN indicates that the energy for the uptake is supplied by oxidation of the substrates through the cytochrome chains and is consistent with Barnes and Kaback's (1) proposals on energy coupling. The transport is also effectively inhibited by the proton conductor CCCP or cation conductors like valinomycin and gramicidin. These compounds consistently inhibit almost all transport systems in the membrane vesicles (1, 2, 9, 15). Their action can be explained by "uncoupling" of transport from oxidation of substrates through the cytochrome chain according to Lombardi et al. (16) or better by the destruction of the potential gradient across the membrane according to Mitchell's (8, 17) chemiosmotic hypothesis. ACKNOWLEDGMENTS This investigation was supported by grant GB 31367 from the National Science Foundation. I thank D. Clark, K. Toth, and K. Farrelly for technical assistance and S. Silver for useful suggestions and helpful criticism during preparation of this manuscript.
LITERATURE CITED 1. Barnes, E. M., Jr., and H. R. Kaback. 1971. Mechanisms of active transport in isolated membrane vesicles. I. The site of energy coupling between D-lactic dehydro-
genase and 6-galactoside transport in Escherichia coli
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membrane vesicles. J. Biol. Chem. 246:5518-5522. 2. Bhattacharyya, P. 1970. Active transport of manganese in isolated membranes of Escherichia coli. J. Bacteriol. 104:1307-1311. 3. Bhattacharyya, P., W. Epstein, and S. Silver. 1971. Valinomycin-induced uptake of potassium in membrane vesicles from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 68:1488-1492. 4. Bhattacharyya, P., L. Wendt, E. Whitney, and S. Silver. 1970. Colicin-tolerant mutants of Escherichia coli: resistance of membranes to colicin El. Science 168:998-1000. 5. Dietz, G. W. 1972. Dehydrogenase activity involved in the uptake of glucose-6-phosphate by a bacterial membrane system. J. Biol. Chem. 247:4561-4565. 6. Eisenstadt, E., S. Fisher, C.-L. Der, and S. Silver. 1973. Manganese transport in Bacillus subtilis W23 during growth and sporulation. J. Bacteriol. 113:1363-1372. 7. Fisher, S., L. Buxbaum, K. Toth, E. Eisenstadt, and S. Silver. 1973. Regulation of the rate of manganese accumulation and exchange in Bacillus subtilis W23. J. Bacteriol. 113:1373-1380. 8. Harold, F. M. 1972. Conservation and transformation of energy by bacterial membranes. Bacteriol. Rev. 36:172-230. 9. Kaback, H. R. 1969. Studies on sugar transport by isolated bacterial membrane preparations, p. 421-441. In D. C. Tosteson (ed.), The molecular basis of membrane function. Prentice-Hall, Inc., Englewood Cliffs, New Jersey. 10. Kaback, H. R. 1971. Bacterial membranes. Methods Enzymol. 22:99-120. 11. Kaback, H. R., and E. M. Barnes, Jr. 1971. Mechanisms of active transport in isolated membrane vesicles. II. The mechanism of energy coupling between D-lactiq, dehydrogenase and ,B-galactoside transport in mem-
12.
13.
14.
15.
16.
17.
18.
19.
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brane preparations from Escherichia coli. J. Biol. Chem. 246:5523-5531. Kaback, H. R., and L. Milner. 1970. Relationship of a membrane-bound D-(-)-lactic dehydrogenase to amino acid transport in isolated bacterial membrane preparations. Proc. Natl. Acad. Sci. U.S.A. 66:1008-1015. Konings, W. N., E. M. Barnes, Jr., and H. R. Kaback. 1971. Mechanisms of active transport in isolated membrane vesicles. III. The coupling of reduced phenazine methosulphate to the concentrative uptake of 6-galactosides and amino acids. J. Biol. Chem. 246:5857-5861. Konings, W. N., A. Bisschop, M. Vennhuis, and C. A. Vermeulen. 1973. A new procedure for the isolation of membrane vesicles of Bacillus subtilis and an electron microscope study of their ultrastructure. J. Bacteriol. 116:1456-1465. Konings, W. N., and E. Freese. 1972. Amino acid transport in membrane vesicles of Bacillus subtilis. J. Biol. Chem. 247:2408-2418. Lombardi, F. J., J. P. Reeves, and H. R. Kaback. 1973. Mechanisms of active transport in isolated bacterial membrane vesicles. XIII. Valinomycin-induced rubidium transport. J. Biol. Chem. 248:3551-3565. Mitchell, P. 1970. Reversible coupling between transport and chemical reactions, p. 192-256. In E. E. Bittar (ed.), Membranes and ion transport, vol. 1. WileyInterscience, New York. Short, S. A., D. C. White, and H. R. Kaback. 1972. Mechanisms of active transport in isolated bacteria! membrane vesicles. IX. The kinetics and specificity of amino acid transport in Staphylococcus aureus membrane vesicles. J. Biol. Chem. 247:7452-7458. Silver, S., P. Johnseine, and K. King. 1970. Manganese active transport in Escherichia coli. J. Bacteriol. 104:1299-1306.
JOURNAL OF BACTERoLOGY, July 1975, p. 123-127 Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Active Transport of Manganese in Isolated Membrane Vesicles of Bacillus subtilis PINAKILAL BHATFACHARYYA' Department of Biology, Washington University, St. Louis, Missouri 63130 Received for publication 14 April 1975
Membrane vesicles isolated from cells of Bacillus subtilis W23 accumulate in the presence of an energy source. The artificial electron donor system ascorbate and phenazine methosulfate or reduced nicotinamide adenine dinucleotide and phenazine methosulfate can supply the energy for the uptake. D-Lactate in the presence or absence of phenazine methosulfate would not support manganese accumulation. Anaerobiosis, cyanide, m-chlorophenyl carbonylcyanide hydrazone, valinomycin, gramicidin, and p-hydroxy-mercuribenzoate inhibit the uptake. The inhibition by p-hydroxymercuribenzoate is prevented by excess dithiothreitol. Potassium fluoride or sodium arsenate has no effect on the uptake. The manganese transport system in the B. subtilis vesicles exhibits Michaelis-Menten kinetics with a Km of 13 gM and a Vmax of 1.7 nmol/min per mg (dry weight) of membranes. The uptake of manganese is specific and is not inhibited by 0.1 mM CaCl2 or MgCl2. manganese
Studies with isolated membrane vesicles have revealed that active transport systems for a number of structurally different compounds including sugars (1, 5, 9, 11), amino acids (4, 12, 13, 15, 18), manganese (2), and potassium (3, 16) reside in the cytoplasmic membranes of different organisms. The transport systems may be coupled to either the phosphoenolpyruvate phosphotransferase system (in the case of glucose and fructose) or to a membrane-bound dehydrogenase (1, 9, 11, 12, 15). The energy substrate for dehydrogenase-coupled transport differs in the membrane vesicles from different organisms depending upon the nature of the organism and conditions of growth. Where Dlactate is the most effective energy source for transport in Escherichia coli and Micrococcus denitrificans membranes (1, 11, 12), transport of amino acids in Staphylococcus aureus membranes is coupled to a-glycerophosphate dehydrogenase (18) and in Bacillus subtilis membranes to reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase or a-glycerophosphate dehydrogenase, if the cells are grown in glycerol (15). The present paper describes an energy-dependent manganese accumulation system in the membrane vesicles from log-phase cells of B. subtilis W23 in the presence of NADH-phenazine methosulfate (PMS) or as' Present address: Marrs McLean Department of Biochemistry, Baylor College of Medicine, Texas Medical Center, Houston, Tex. 77025.
123
corbate-PMS. The corresponding transport system in the whole cells of B. subtilis has been recently reported from this laboratory (6, 7). MATERIALS AND METHODS Organism and media. B. subtilis strain W23 was used. Cells were maintained on tryptone agar and grown in tryptone broth (6) or in tryptone broth supplemented with 25 mM KCl and CaCl2 and MgCl2, each 1.0 mM. Preparation of membrane vesicles. In early experiments, vesicles were prepared by the method of Kaback (10) as modified and described by Konings and Freese (15). Later, the membrane vesicles were prepared by a modified and simpler method as developed and communicated by Konings et al. (14). Cells were harvested from mid-log phase of growth and washed twice with 0.1 M K-PO4 buffer (pH 7.3). It is important to harvest the cells before they reach the stationary phase of growth since stationary phase cells give inactive or defective vesicles. The pellet was then resuspended thoroughly in 50 mM K-PO4 (pH 8.0) buffer and diluted into a large volume of 50 mM K-PO4 buffer (pH 8.0) containing 300 Mg of lysozyme per ml, 10 Asg of deoxyribonuclease per ml, 10 Ag of ribonuclease per ml, and 10 mM MgSO4. The volume of this incubation mixture was approximately 300 mVg (wet weight) of cells or at least one-third of the original growth medium volume. The diluted cells were then incubated for 45 min at 37 C. Ethylenediaminetetraacetate (K-salt, adjusted to pH 8.0 with KOH) was then added to 15 mM final concentration, and the mixture was further incubated for 15 min, followed by an additional 30-min incubation after the addition of 20 mM MgSO4. Cells, by now converted
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into vesicles, were then centrifuged at 16,000 x g for 30 to 45 min at 4 C. The pellet was washed twice with 0.1 M K-PO4 buffer (pH 6.6) with repeated homogenization and centrifugation. If the pellet was sticky, it was suspended in 50 ml of 0.1 M K-PO4 buffer (pH 6.6) containing 10 ,gg each of deoxyribonuclease and ribonuclease per ml and 10 mM MgSO4 and incubated for 30 min with stirring. Before collecting the final pellet at 34,000 x g for 30 min, two low-speed contrifugation pellets (800 x g) were discarded. The 34,000 x g pellet was then suspended in 0.25 M sucrose by homogenization and again centrifuged. The vesicles were then resuspended in 0.25 M sucrose to an approximate Klett reading of 700 (ca. 540 nm, green filter), frozen in 1.0- or 0.5-ml portions in a dry ice-acetone bath, and stored at -80 C. Manganese uptake by B. subtilis vesicles. Frozen vesicles were thawed at 30 C and diluted with 0.25 M sucrose. Appropriate amounts were then distributed into several disposable culture tubes (10 by 75 mm), and additions of different substances were made so that the final volume of the mixture was 0.1 ml and the composition was as described in the figure legends. During incubation, the tubes were placed in a water bath over a magnetic stirrer and stirred with Teflon-coated 7-mm magnets while either 0, or Nwas blown over the surface of the incubation mixture. At indicated times the tubes were taken out of the water bath, and the contents of the tubes were diluted with 2 ml of 0.25 M LiCl and filtered through membrane filters (type HA; Millipore Corp., Bedford, Mass.) and washed further with 2 ml of 0.25 M LiCl. Washing with LiCl gives optimum results with transport studies in membrane vesicles (1), and 0.25 M LiCl gives optimum results for overall manganese uptake under the experimental conditions. The radioactivity on the filters was then determined in a scintillation spectrometer as described earlier (19). Each point was corrected for a background obtained by diluting the contents of a control tube with LiCl before the addition of the radioactive manganese chloride. Uptake is expressed as nanomoles of manganese taken up per milligram (dry weight) of membranes. Materials. Reagent grade chemicals and deionized water were used in all experiments. D-(- )-Lactic acid (lithium salt), NADH (disodium salt), p-chloromercuribenzoate (sodium salt), and PMS were purchased from Sigma Chemical Co., St. Louis, Mo.; m-chlorophenyl carbonylcyanide hydrazone (CCCP) and valinomycin from Calbiochem, Los Angeles, Calif.; dithiothreitol from P. L. Biochemicals, Inc., Milwaukee, Wis.; and gramicidin from Mann Research Laboratory, New York, N.Y. Radioactive [4C Jproline and carrier-free radioactive 54MnCl, were purchased from New England Nuclear Corp., Boston, Mass.
PMS and oxygen were blown over the surface of the mixture. The addition of radioactive 54MnCl, led to rapid accumulation of the radioactivity inside the vesicles that reached a maximum at 10 min (Fig. 1). On further incubation, the accumulated manganese was released from the vesicles presumably after the exhaustion of the energy source (15). The maximal level of uptake at about 10 min does not represent cessation of manganese uptake altogether but an equilibrium of influx and efflux. Thus when the membranes were incubated with nonradioactive 10-6 M manganese chloride and a trace amount of radioactive 54MnCl, was added at 10 min, there was further uptake of radioactivity but with a lower rate and lower equilibrium level than when 54MnCl, was added at the beginning. Effects of energy substrates and metabolic inhibitors. Under anaerobic conditions, there was very little uptake of manganese by the B. subtilis vesicles (Fig. 2A). Omission of either PMS or ascorbate resulted in manganese accumulation no higher than that observed under anaerobic conditions. NADH did not support manganese uptake to any significant extent in the absence of PMS (data not shown), but in the presence of PMS the rate of manganese uptake was almost the same with either NADH or ascorbate. There was no uptake with D-lactate in the presence (Fig. 2A) or absence of PMS
RESULTS Accumulation of manganese by membranes. When the vesicles were suspended in 0.2 M sucrose in the presence of an artificial electron donor system, sodium ascorbate and
FIG. 1. Manganese accumulation by Bacillus subtilis membranes. The incubation medium contained 0.2 M sucrose, 20 mM sodium ascorbate, 0.1 mM PMS, and approximately 2 mg of membranes per ml. Nonradioactive 10- M MnCl, was added at 0 min and trace 54MnCI, was added at 0 min (0) or 10 min (0).
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FIG. 2. Effect of energy substrates and metabolic inhibitors on the manganese uptake. (A) The incubation medium contained 0.2 M sucrose, 10 mM K-PO4 buffer (pH 6.6), 20 mM energy substrates, 0.1 mM PMS, and 3 mg of membranes per ml. "4MnCl, was added at 10- 6M. Symbols: 0, ascorbate + PMS + 0,; 0, ascorbate + PMS + N,; A, NADH + PMS + 0,, A, D-lactate + PMS + O;, 0, PMS + 0 - ascorbate; M, ascorbate + 0° - PMS. (B) The incubation mixture contained 0.2 M sucrose, 10 mM K-PO4 buffer (pH 6.6), 1 mM MgSO4, 20 mM sodium ascorbate, 0.1 mM PMS, and 3.3 mg of membranes per ml. Inhibitors were added immediately prior to the addition of "'MnCI,. Symbols: 0, control; 0, 10-6 M CCCP; A, 10-' M p-hydroxymercuribenzoate; A, 10-' M p-hydroxymercuribenzoate + 5 x 10-4 M dithiothreitol; 0, 10-2 M KCN; *, 2 x 10-6 M valinomycin; V, 2 x 10- 6 Mgramicidin.
(data not shown). Thus the energy for manganese accumulation by B. subtilis can be supplied by the oxidation of reduced PMS or NADH through the cytochrome chain. Figure 2B shows the effects of metabolic poisons on manganese uptake by the B. subtilis membranes. Potassium cyanide, an inhibitor of electron transport, inhibited manganese accumulation almost completely. A sulfhydryl reagent, p-hydroxymercuribenzoate, when added just prior to the addition of the radioactive 54MnCl2, inhibited the uptake about 40%, and the inhibition was antagonized by prior addition of 5 x 10-8 M dithiothreitol. Potassium fluoride (10-2 M) or sodium arsenate (1.6 x 10-2 M) did not affect the manganese uptake to any significant extent (data not shown). The proton conductor CCCP or ionophores like valinomycin and gramicidin inhibited manganese uptake very effectively (Fig. 2B). Kinetics of minganese transport. The initial rate of manganese uptake was determined in the presence of varying concentrations of
125
manganese (Fig. 3). Over the range from 1 to 24 ,M Mn2 , the manganese uptake in the vesicles showed Michaelis-Menten kinetics with a Km of 13 gM and Vmax of 1.7 nmol/min per mg of membranes. However, increasing the concentration of the substrate above 25 ,M, up to 200 ,M, never resulted in complete saturation of the system for manganese accumulation. Specificity of the manganese uptake. Near the micromolar range of manganese, the system is highly specific, and manganese uptake is not inhibited by magnesium or calcium (Fig. 4). When the incubation medium was made using either Na+ or K+ salts of ascorbate, addition of K+ or Na+ salts up to 10 mM concentration did not significantly alter the uptake of manganese (data not shown). However, ferrous chloride and cuprous chloride at 0.1 mM concentrations seriously inhibit the accumulation of radioactive manganese. The inhibition by these salts is likely due to inhibition of the energy metabolism in the membranes or to some nonspecific changes in the membranes, since proline uptake is also inhibited to a comparable extent (Fig. 4B). The values for proline uptake in the vesicles are considerably lower than those reported by Konings and Freese (15). Since the vesicles were prepared for the purpose of studying manganese uptake, they were washed and stored in sucrose. Proline uptake by such vesicles is poor.
/
1000
rE
~~~~~~~~0
0.004 ~~~~~0
1 * 0
5 0.003 a 0.02
0
j 0
8
16
24
[Manganesel [#M]
FIG. 3. Kinetics of manganese accumulation. Incubation medium contained 0.2 M sucrose, 10 mM tris(hydroxymethylaminomethane-hydrochloride (pH 7.0), 20 mM sodium ascorbate, 0.1 mM PMS, and 3 mg of membranes per ml. Temperature of incubation was 30 C, and the initial rates of uptake were determined over the first minute after the addition of
"4MnCI2.
126
BHATTACHARYYA
J. BACTROL.
u2
0
+ ~ 0 +C
~
~
~
O
O
1
200
O~~~~~~~~~~~~~~~~~~O
2
3
0
2
4
minutes at 30 C
FIG. 4. Effect of cation on the uptake of manganese and proline. (A) Membranes were suspended at I mg/ml in 0.2 M sucrose, 20 mM sodium ascorbate, and 0. 1 mM PMS. 114MnCl, was added at 10- " M and nonradioactive salts at 10-4 M. Symbols: O,
control; *, CaCI,,, A, MgCI,, A, FeC1, O, CuCI, *,
MnCl.. (B) The incubation medium contained 0.2 M sucrose, 20 mM sodium ascorbate, 0.1 mM PMS, 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.0) and 1.3 mg of membranes per ml. Rad ioactive 1lm4Clproline was 2.4t added at M. DISCUSSION Manganese uptake in membrane vesicles of B. subtilis showed qualitatively similar properties to manganese active transport in intact cells (6), for example, requirement for energy metabolism, saturation kinetics, and substrate specificity. Similar observations were reported on manganese accumulation in membrane vesicles of E. coli (2). However, with respect to manganese transport, membrane vesicles of E. coli differ from those of B. subtilis in one significant aspect. Manganese uptake in E. coli membranes is dependent on the presence of CaClb in the incubation medium, whereas that in B. subtilis membranes is not. The kinetics studies of the manganese uptake in the B. subtilis membrane vesicles yielded a Kc of approximately 13 BM and Vsax of 17 nmol/min per mg of membranes. The corresponding values for the whole cells are 1 sM and 0.5 nmol/min per mg (6). Calculating on a rough basis, the Vax in the membrane vesicles is about one-third of that in the wholescells and compares favorably with similar determinations in other transport systems (2, 9, 11). The higher value for the KVmwith membrane vesicles does not mean that manganese transport by the membranes is significantly different from that
by the intact cells, since changes in cell wall shielding components would be expected to influence trace, low Vmax systems such as that for manganese especially strongly. Alternatively, components of the system may be altered during membrane preparation. Most of the accumulated manganese does not appear to be free, since adding trace 54MnCl2 after 10-min incubation with 10-6 M nonradioactive MnCl2 resulted in a lower equilibrium value for "4Mn uptake than that obtained when the radioactivity was added at the beginning of the incubation (Fig. 1). Furthermore, addition of excess nonradioactive manganese chloride or the uncoupling agent CCCP at any point during the active uptake of "4MnCl2 resulted in an immediate cessation of uptake of radioactivity but very little release of already accumulated radioactivity (data not shown). With levels of accumulation in the picomoles per milligram range, absorption to intramembrane polyanions could account for the lack of extensive exchange. The energy for the manganese uptake in the membrane vesicles from B. subtilis could be supplied by NADH and PMS or ascorbate and PMS but not by D-lactate. This confirms the observations of Konings and Freese (15) on energy sources for amino acid transport in B. subtilis membrane. The inhibition of the manganese uptake by anaerobiosis and KCN indicates that the energy for the uptake is supplied by oxidation of the substrates through the cytochrome chains and is consistent with Barnes and Kaback's (1) proposals on energy coupling. The transport is also effectively inhibited by the proton conductor CCCP or cation conductors like valinomycin and gramicidin. These compounds consistently inhibit almost all transport systems in the membrane vesicles (1, 2, 9, 15). Their action can be explained by "uncoupling" of transport from oxidation of substrates through the cytochrome chain according to Lombardi et al. (16) or better by the destruction of the potential gradient across the membrane according to Mitchell's (8, 17) chemiosmotic hypothesis. ACKNOWLEDGMENTS This investigation was supported by grant GB 31367 from the National Science Foundation. I thank D. Clark, K. Toth, and K. Farrelly for technical assistance and S. Silver for useful suggestions and helpful criticism during preparation of this manuscript.
LITERATURE CITED 1. Barnes, E. M., Jr., and H. R. Kaback. 1971. Mechanisms of active transport in isolated membrane vesicles. I. The site of energy coupling between D-lactic dehydro-
genase and 6-galactoside transport in Escherichia coli
VOL. 123, 1975
MANGANESE TRANSPORT IN BACILLUS MEMBRANES
membrane vesicles. J. Biol. Chem. 246:5518-5522. 2. Bhattacharyya, P. 1970. Active transport of manganese in isolated membranes of Escherichia coli. J. Bacteriol. 104:1307-1311. 3. Bhattacharyya, P., W. Epstein, and S. Silver. 1971. Valinomycin-induced uptake of potassium in membrane vesicles from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 68:1488-1492. 4. Bhattacharyya, P., L. Wendt, E. Whitney, and S. Silver. 1970. Colicin-tolerant mutants of Escherichia coli: resistance of membranes to colicin El. Science 168:998-1000. 5. Dietz, G. W. 1972. Dehydrogenase activity involved in the uptake of glucose-6-phosphate by a bacterial membrane system. J. Biol. Chem. 247:4561-4565. 6. Eisenstadt, E., S. Fisher, C.-L. Der, and S. Silver. 1973. Manganese transport in Bacillus subtilis W23 during growth and sporulation. J. Bacteriol. 113:1363-1372. 7. Fisher, S., L. Buxbaum, K. Toth, E. Eisenstadt, and S. Silver. 1973. Regulation of the rate of manganese accumulation and exchange in Bacillus subtilis W23. J. Bacteriol. 113:1373-1380. 8. Harold, F. M. 1972. Conservation and transformation of energy by bacterial membranes. Bacteriol. Rev. 36:172-230. 9. Kaback, H. R. 1969. Studies on sugar transport by isolated bacterial membrane preparations, p. 421-441. In D. C. Tosteson (ed.), The molecular basis of membrane function. Prentice-Hall, Inc., Englewood Cliffs, New Jersey. 10. Kaback, H. R. 1971. Bacterial membranes. Methods Enzymol. 22:99-120. 11. Kaback, H. R., and E. M. Barnes, Jr. 1971. Mechanisms of active transport in isolated membrane vesicles. II. The mechanism of energy coupling between D-lactiq, dehydrogenase and ,B-galactoside transport in mem-
12.
13.
14.
15.
16.
17.
18.
19.
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brane preparations from Escherichia coli. J. Biol. Chem. 246:5523-5531. Kaback, H. R., and L. Milner. 1970. Relationship of a membrane-bound D-(-)-lactic dehydrogenase to amino acid transport in isolated bacterial membrane preparations. Proc. Natl. Acad. Sci. U.S.A. 66:1008-1015. Konings, W. N., E. M. Barnes, Jr., and H. R. Kaback. 1971. Mechanisms of active transport in isolated membrane vesicles. III. The coupling of reduced phenazine methosulphate to the concentrative uptake of 6-galactosides and amino acids. J. Biol. Chem. 246:5857-5861. Konings, W. N., A. Bisschop, M. Vennhuis, and C. A. Vermeulen. 1973. A new procedure for the isolation of membrane vesicles of Bacillus subtilis and an electron microscope study of their ultrastructure. J. Bacteriol. 116:1456-1465. Konings, W. N., and E. Freese. 1972. Amino acid transport in membrane vesicles of Bacillus subtilis. J. Biol. Chem. 247:2408-2418. Lombardi, F. J., J. P. Reeves, and H. R. Kaback. 1973. Mechanisms of active transport in isolated bacterial membrane vesicles. XIII. Valinomycin-induced rubidium transport. J. Biol. Chem. 248:3551-3565. Mitchell, P. 1970. Reversible coupling between transport and chemical reactions, p. 192-256. In E. E. Bittar (ed.), Membranes and ion transport, vol. 1. WileyInterscience, New York. Short, S. A., D. C. White, and H. R. Kaback. 1972. Mechanisms of active transport in isolated bacteria! membrane vesicles. IX. The kinetics and specificity of amino acid transport in Staphylococcus aureus membrane vesicles. J. Biol. Chem. 247:7452-7458. Silver, S., P. Johnseine, and K. King. 1970. Manganese active transport in Escherichia coli. J. Bacteriol. 104:1299-1306.
