Vol. 123, No. 3 Printed in U.S.A.
OF BACTERIOLOGY, Sept. 1975, p. 985-991 Copyright 0 1975 American Society for Microbiology
JOURNAL
Energy Coupling for Methionine Transport in Escherichia coli ROBERT J. KADNER* AND HERBERT H. WINKLER Department of Microbiology, The University of Virginia School of Medicine, Charlottesville, Virginia 22901 Received for publication 12 June 1975
The source of metabolic energy for the accumulation of methionine in cells of Escherichia coli was shown to differ from that for proline uptake. In contrast to proline uptake, methionine accumulation was sensitive to arsenate, and relatively resistant to azide or dinitrophenol. Adenosine triphosphatase mutant strains also differentiated between the two systems, consistent with the conclusion that, although proline uptake is driven directly by the energized membrane state, methionine uptake is not. Methionine transport is similar to that of other osmotic shock-sensitive systems in its direct utilization of adenosine 5'-triphosphate or a related compound as energy source.
Recently, evidence has been accumulating for the existence of two discrete mechanisms of energy coupling for active transport in cells of Escherichia coli. One of these mechanisms is that found to be operative for a large number of transport systems in the bacterial membrane vesicle preparations studied by Kaback and his colleagues (9). Transport is driven by an energized state of the membrane, which can be generated by electron transport. The identity of this energized state is still in question. Attempts to identify it with a proton-motive force in accord with a chemiosmotic model are receiving support, although some results are difficult to explain under this model (10). This energy coupling mechanism for these transport systems also appears to operate in whole cells (13). On the other hand, Berger and Heppel (1, 2) have proposed that glutamine transport, a process observed in cells but not in vesicles, can only utilize energy directly from adenosine 5'-triphosphate (ATP) or a related compound rather than the energized membrane state. This ATPdriven process has subsequently been shown for a number of other transport systems, including those for galactose (20), ribose (5) glycylglycine (4), isoleucine, arginine, histidine, ornithine, and ac-diaminopimelic acid (2). All of these transport systems also share the characteristic of sensitivity to osmotic shock, associated in most cases with the release of specific binding proteins. A major portion of the evidence for the nature of the two mechanisms of energy coupling comes from studies of mutants in the Ca2+- and Mg2+-dependent membrane adenosine triphosphatase (ATPase). The role of this enzyme appears to be the reversible formation of ATP at
the expense of energy of the energized membrane state (3). With these mutants, it is possible to dissociate the energized membrane state from the level of ATP. This paper presents evidence that the transport systems for methionine, which are sensitive to osmotic shock, are driven by phosphate bond energy, presumably ATP. This evidence includes the effect of several inhibitors on uptake into cells, as well as the effect of dissociating cellular ATP levels from the energized membrane state through the use of mutants lacking heme (i.e., electron transport) or the ATPase. Membrane vesicle preparations lack methionine transport activity (14). MATERIALS AND METHODS Bacterial strains and growth. E. coli strain 7 was used as the wild-type strain for most of these studies. Strain 7 and NR70 (an isogenic derivative lacking the Ca2+,Mg2+-ATPase [17]) were obtained from B. Rosen. The isogenic pair DG1 (uncA+) and N144 (uncA) of Gutnick et al. (8) and the uncA strain AN 120 (3) were provided by H. R. Kaback. The hemA mutant RK4303 was isolated from Lin's strain E15 as a spontaneous neomycin-resistant mutant that was dependent on the addition of S-aminolevulinic acid (ALA) for growth on oxidative carbon sources, such as succinate. The mutation was 20% co-transducible with chIC. Cultures were grown in the basal salts medium A of Davis and Mingioli (6) supplemented with 0.5% glucose, thiamine (1 tg/ml), and any required amino acids (100 ug/ml). Strain RK4303 was grown aerobically in a medium containing ALA (20 ,g/ml), and anaerobically in the absence of ALA. All transport assays with this strain were performed aerobically. After their incubation at 37 C in a gyratory shaker, cultures were routinely harvested in mid-exponential phase (6 x 101 to 8 x 10" cells/ml) by centrifugation. Transport assays. The intention of this paper is to 985
986
J . BACTrERIOL.
KADNER AND WINKLER
demonstrate that the energized membrane state, which drives proline uptake, is not responsible for the accumulation of methionine by whole cells. It is generally accepted that proline uptake is driven by the energized membrane state. To facilitate the comparison of the energetics of the transport of these two amino acids, the transport of both was simultaneously determined in the same assay. For this purpose, the transport substrate was an equimolar mixture of L- [JH Imethionine and L- [14C ]proline, adjusted so that each amino acid provided approximately the same radioactivity in the assay mixture. For the transport assays, the cells were washed twice by centrifugation at 4 C in medium A containing chloramphenicol (100 gg/ml) and glucose (0.5%) unless indicated otherwise. The cells were resuspended in the same medium to a cell density of 1.6 x 109 per ml and maintained at 23 C. Unless specified, all assays were incubated at 23 C and were completed within 2 h from the time of harvest. For experiments on the effect of arsenate, cells were washed twice and resuspended in 90 mM KCl, containing either 10 mM potassium phosphate (pH 7.0) or 10 mM potassium arsenate (pH 7.0), and incubated in these media for 15 min before assay. The methods for transport assays have been described (11). Usually three samples were collected within 1.5 min from the time of mixing the cells and the substrate, and the uptake is usually expressed as picomoles of substrate accumulated in 0.5 min per microliter of cell water. Multiplication of these values by 0.004 yields nanomoles per milligram of cell protein. Starvation or osmotic shock of cells were carried out essentially as described by Berger (1) or Weiner and Heppel (19), respectively. Starvation with 2,4dinitrophenol (DNP) (5 mM) was for 10 h with uncA + strains and 1 h for uncA strains. The cells were starved by aeration at 37 C in medium A containing DNP. After the starvation period, the cells were washed twice with medium A and assayed for transport within 2 h. When indicated, energy sources were added to the cells for 10 min before the addition of the labeled substrates. Chemicals. The radioactive compounds were purchased from New England Nuclear Corp. Most of the other chemicals were obtained from Sigma Chemical Co.
RESULTS It had been previously noted that the accumulation of methionine was partially inhibited by the presence of either the inhibitor of glycolysis, fluoride, or the inhibitors of oxidative energy generation, azide, dinitrophenol, arsenite, or carbonyl-cyanide m-chlorophenylhydrazone (CCCP) (11). Both types of inhibitors had to be present for full inhibition of uptake. It was proposed then that methionine uptake was driven by some metabolic product common to both glycolysis and oxidative metabolism, possibly ATP. It has also been shown that both L-methionine uptake (12) and the D-methio-
nine uptake system (unpublished data) exhibited essentially identical responses to these various inhibitors, so that even though both systems contribute to the observed uptake, they both respond similarly to the energy supply. Effects of arsenate on transport. Incubation of cells of strain 7 in 10 mM arsenate with 90 mM KCl for 15 min reduced glucose-driven methionine uptake by about 75% (Table 1). These cells still effectively transport proline at levels ranging from 75 to 100% of the rate of cells incubated with phosphate. The inhibition of methionine uptake in arsenate-treated cells could be reversed to a large extent by addition of 10 mM phosphate; the decrease in proline uptake was not appreciably affected by this treatment. Methionine transport in the ATPaseless strain NR70 was also reduced greater than 80% by arsenate. Glucose-driven proline transport in this strain was inhibited by arsenate to a greater degree than in the wild-type strain. This inhibition of methionine uptake by arsenate suggests that a phosphorylated product (probably ATP) can drive methionine transport.
Transport of Ca2+,Mg2+-ATPase-deficient mutants. The Ca2+, Mg2+-ATPase is necessary for the synthesis of ATP by oxidative phosphorylation; hence mutant strains lacking this activity are unable to grow with a nonfermentable carbon source, such as succinate. The transport properties of three independent ATPase mutants were determined with unstarved cells in the presence of glucose and several inhibitors (Table 2). The parental strains, 7 and DG1, differed somewhat with respect to the susceptibility of methionine transport to uncouplers, but otherwise showed similar properties. Two of the mutants, N144 and AN120, possessed rates of methionine and proline uptake similar to those in wild-type strains. Methionine uptake in these strains was more susceptible to inhibition by fluoride and significantly less susceptible to inhibition by uncouplers, as described later. In TABLE 1. Inhibition of methionine transport by arsenate Substrate accumulated (pmol/Ml in Strain
1 min)
Medium Methio-
7
NR70
Phosphate Arsenate Phosphate Arsenate
nine 116
Proline 114
26
103
83 15
29 19
ENERGETICS OF METHIONINE TRANSPORT
VOL. 123, 1975
TABLE 2. Transport in several Ca2+,Mg2+-ATPase mutants and the effect of NaF, DNP, and CCCPa Strain
7
DG1
Addition
138 80 (58) 20.5 (15) 20.5(15)
None NaF DNP
158 84 (53) 69 (44) 53 (34)
84 57 (67)
130 39 (30) 93 (71) 64 (49)
90 89 2.5 1
CCCP
113 36 (32) 40 (35) 46.3 (41)
78 66 (85) 1 1
None NaF DNP CCCP
93 8 (8) 47 (51) 34 (36)
None NaF DNP
CCCP AN 120
None NaF DNP
NR 70
117 56 (48) 7.5 (6)
None NaF DNP CCCP
CCCP N144
Substrate accumulated (pmol/ul at 1 min) Proline Methionine
1(
OF BACTERIOLOGY, Sept. 1975, p. 985-991 Copyright 0 1975 American Society for Microbiology
JOURNAL
Energy Coupling for Methionine Transport in Escherichia coli ROBERT J. KADNER* AND HERBERT H. WINKLER Department of Microbiology, The University of Virginia School of Medicine, Charlottesville, Virginia 22901 Received for publication 12 June 1975
The source of metabolic energy for the accumulation of methionine in cells of Escherichia coli was shown to differ from that for proline uptake. In contrast to proline uptake, methionine accumulation was sensitive to arsenate, and relatively resistant to azide or dinitrophenol. Adenosine triphosphatase mutant strains also differentiated between the two systems, consistent with the conclusion that, although proline uptake is driven directly by the energized membrane state, methionine uptake is not. Methionine transport is similar to that of other osmotic shock-sensitive systems in its direct utilization of adenosine 5'-triphosphate or a related compound as energy source.
Recently, evidence has been accumulating for the existence of two discrete mechanisms of energy coupling for active transport in cells of Escherichia coli. One of these mechanisms is that found to be operative for a large number of transport systems in the bacterial membrane vesicle preparations studied by Kaback and his colleagues (9). Transport is driven by an energized state of the membrane, which can be generated by electron transport. The identity of this energized state is still in question. Attempts to identify it with a proton-motive force in accord with a chemiosmotic model are receiving support, although some results are difficult to explain under this model (10). This energy coupling mechanism for these transport systems also appears to operate in whole cells (13). On the other hand, Berger and Heppel (1, 2) have proposed that glutamine transport, a process observed in cells but not in vesicles, can only utilize energy directly from adenosine 5'-triphosphate (ATP) or a related compound rather than the energized membrane state. This ATPdriven process has subsequently been shown for a number of other transport systems, including those for galactose (20), ribose (5) glycylglycine (4), isoleucine, arginine, histidine, ornithine, and ac-diaminopimelic acid (2). All of these transport systems also share the characteristic of sensitivity to osmotic shock, associated in most cases with the release of specific binding proteins. A major portion of the evidence for the nature of the two mechanisms of energy coupling comes from studies of mutants in the Ca2+- and Mg2+-dependent membrane adenosine triphosphatase (ATPase). The role of this enzyme appears to be the reversible formation of ATP at
the expense of energy of the energized membrane state (3). With these mutants, it is possible to dissociate the energized membrane state from the level of ATP. This paper presents evidence that the transport systems for methionine, which are sensitive to osmotic shock, are driven by phosphate bond energy, presumably ATP. This evidence includes the effect of several inhibitors on uptake into cells, as well as the effect of dissociating cellular ATP levels from the energized membrane state through the use of mutants lacking heme (i.e., electron transport) or the ATPase. Membrane vesicle preparations lack methionine transport activity (14). MATERIALS AND METHODS Bacterial strains and growth. E. coli strain 7 was used as the wild-type strain for most of these studies. Strain 7 and NR70 (an isogenic derivative lacking the Ca2+,Mg2+-ATPase [17]) were obtained from B. Rosen. The isogenic pair DG1 (uncA+) and N144 (uncA) of Gutnick et al. (8) and the uncA strain AN 120 (3) were provided by H. R. Kaback. The hemA mutant RK4303 was isolated from Lin's strain E15 as a spontaneous neomycin-resistant mutant that was dependent on the addition of S-aminolevulinic acid (ALA) for growth on oxidative carbon sources, such as succinate. The mutation was 20% co-transducible with chIC. Cultures were grown in the basal salts medium A of Davis and Mingioli (6) supplemented with 0.5% glucose, thiamine (1 tg/ml), and any required amino acids (100 ug/ml). Strain RK4303 was grown aerobically in a medium containing ALA (20 ,g/ml), and anaerobically in the absence of ALA. All transport assays with this strain were performed aerobically. After their incubation at 37 C in a gyratory shaker, cultures were routinely harvested in mid-exponential phase (6 x 101 to 8 x 10" cells/ml) by centrifugation. Transport assays. The intention of this paper is to 985
986
J . BACTrERIOL.
KADNER AND WINKLER
demonstrate that the energized membrane state, which drives proline uptake, is not responsible for the accumulation of methionine by whole cells. It is generally accepted that proline uptake is driven by the energized membrane state. To facilitate the comparison of the energetics of the transport of these two amino acids, the transport of both was simultaneously determined in the same assay. For this purpose, the transport substrate was an equimolar mixture of L- [JH Imethionine and L- [14C ]proline, adjusted so that each amino acid provided approximately the same radioactivity in the assay mixture. For the transport assays, the cells were washed twice by centrifugation at 4 C in medium A containing chloramphenicol (100 gg/ml) and glucose (0.5%) unless indicated otherwise. The cells were resuspended in the same medium to a cell density of 1.6 x 109 per ml and maintained at 23 C. Unless specified, all assays were incubated at 23 C and were completed within 2 h from the time of harvest. For experiments on the effect of arsenate, cells were washed twice and resuspended in 90 mM KCl, containing either 10 mM potassium phosphate (pH 7.0) or 10 mM potassium arsenate (pH 7.0), and incubated in these media for 15 min before assay. The methods for transport assays have been described (11). Usually three samples were collected within 1.5 min from the time of mixing the cells and the substrate, and the uptake is usually expressed as picomoles of substrate accumulated in 0.5 min per microliter of cell water. Multiplication of these values by 0.004 yields nanomoles per milligram of cell protein. Starvation or osmotic shock of cells were carried out essentially as described by Berger (1) or Weiner and Heppel (19), respectively. Starvation with 2,4dinitrophenol (DNP) (5 mM) was for 10 h with uncA + strains and 1 h for uncA strains. The cells were starved by aeration at 37 C in medium A containing DNP. After the starvation period, the cells were washed twice with medium A and assayed for transport within 2 h. When indicated, energy sources were added to the cells for 10 min before the addition of the labeled substrates. Chemicals. The radioactive compounds were purchased from New England Nuclear Corp. Most of the other chemicals were obtained from Sigma Chemical Co.
RESULTS It had been previously noted that the accumulation of methionine was partially inhibited by the presence of either the inhibitor of glycolysis, fluoride, or the inhibitors of oxidative energy generation, azide, dinitrophenol, arsenite, or carbonyl-cyanide m-chlorophenylhydrazone (CCCP) (11). Both types of inhibitors had to be present for full inhibition of uptake. It was proposed then that methionine uptake was driven by some metabolic product common to both glycolysis and oxidative metabolism, possibly ATP. It has also been shown that both L-methionine uptake (12) and the D-methio-
nine uptake system (unpublished data) exhibited essentially identical responses to these various inhibitors, so that even though both systems contribute to the observed uptake, they both respond similarly to the energy supply. Effects of arsenate on transport. Incubation of cells of strain 7 in 10 mM arsenate with 90 mM KCl for 15 min reduced glucose-driven methionine uptake by about 75% (Table 1). These cells still effectively transport proline at levels ranging from 75 to 100% of the rate of cells incubated with phosphate. The inhibition of methionine uptake in arsenate-treated cells could be reversed to a large extent by addition of 10 mM phosphate; the decrease in proline uptake was not appreciably affected by this treatment. Methionine transport in the ATPaseless strain NR70 was also reduced greater than 80% by arsenate. Glucose-driven proline transport in this strain was inhibited by arsenate to a greater degree than in the wild-type strain. This inhibition of methionine uptake by arsenate suggests that a phosphorylated product (probably ATP) can drive methionine transport.
Transport of Ca2+,Mg2+-ATPase-deficient mutants. The Ca2+, Mg2+-ATPase is necessary for the synthesis of ATP by oxidative phosphorylation; hence mutant strains lacking this activity are unable to grow with a nonfermentable carbon source, such as succinate. The transport properties of three independent ATPase mutants were determined with unstarved cells in the presence of glucose and several inhibitors (Table 2). The parental strains, 7 and DG1, differed somewhat with respect to the susceptibility of methionine transport to uncouplers, but otherwise showed similar properties. Two of the mutants, N144 and AN120, possessed rates of methionine and proline uptake similar to those in wild-type strains. Methionine uptake in these strains was more susceptible to inhibition by fluoride and significantly less susceptible to inhibition by uncouplers, as described later. In TABLE 1. Inhibition of methionine transport by arsenate Substrate accumulated (pmol/Ml in Strain
1 min)
Medium Methio-
7
NR70
Phosphate Arsenate Phosphate Arsenate
nine 116
Proline 114
26
103
83 15
29 19
ENERGETICS OF METHIONINE TRANSPORT
VOL. 123, 1975
TABLE 2. Transport in several Ca2+,Mg2+-ATPase mutants and the effect of NaF, DNP, and CCCPa Strain
7
DG1
Addition
138 80 (58) 20.5 (15) 20.5(15)
None NaF DNP
158 84 (53) 69 (44) 53 (34)
84 57 (67)
130 39 (30) 93 (71) 64 (49)
90 89 2.5 1
CCCP
113 36 (32) 40 (35) 46.3 (41)
78 66 (85) 1 1
None NaF DNP CCCP
93 8 (8) 47 (51) 34 (36)
None NaF DNP
CCCP AN 120
None NaF DNP
NR 70
117 56 (48) 7.5 (6)
None NaF DNP CCCP
CCCP N144
Substrate accumulated (pmol/ul at 1 min) Proline Methionine
1(
