Vol. 174, No. 23
JOURNAL OF BACrERIOLOGY, Dec. 1992, p. 7579-7584
0021-9193/92/237579-06$02.00/0 Copyright e 1992, American Society for Microbiology
Mechanism of Glutamate Uptake in Zymomonas mobilis JUTrA RUHRMANN AND REINHARD KRAMER* Institut fiir Biotechnologie I, Forschungszentrum Julich, Postfach 1913, 5170 Julich, Germany Received 17 August 1992/Accepted 4 October 1992
The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanolproducing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of3.5 to 4 mM. Because ofeffective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed ofa pH gradient of0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high V. of 40 ,umol min1 g (dry mass) of celis- and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.
contained (per liter) 50 g of glucose, 10 g of Bacto yeast extract, 1 g of KH2PO4, 1 g of (NH4)2SO4, and 0.5 g of MgSO4. 7H20 (pH 5.0). For uptake experiments, cells were harvested at a cell density of 0.35 to 0.5 mg (dry mass) per ml. The cells were washed twice in buffer containing 100 mM MES (morpholineethanesulfonic acid), 10 mM NaCl, 10 mM KCI, and 3 to 5% glucose and suspended in the same buffer at a cell density of 2.1 to 2.4 mg (dry mass) per ml. If not mentioned otherwise, 3 mM aminooxyacetic acid, an inhibitor of aminotransferases, was added. For investigating the inducibility of the glutamate uptake system, cells were grown in mineral medium, modified from that described in reference 16, which contained (per liter) 1.0 g of MgSO4- 7H20, 3.48 g of KH2PO4, 0.2 g of citric acid monohydrate, 0.01 g of FeSO4. 7H20, 19.52 g of MES, and 4.4 g of glutamate (30 mM) or 1.98 g of (NH4)2SO4 as an N source (pH 5.8). After sterilization, calcium pantothenate and biotin were added to a final concentrations of 1.5 mg of each per liter. Chemicals. Radiochemicals were purchased from Amersham International (Amersham, Buckinghamshire, United Kinlgdom). The following labelled compounds were used: [U-'4C]glutamic acid, [U-14C]tetraphenylphosphonium bromide, [U-14Ctaurine, 2-[1494methylamine, [U-14C]benzoic acid, 3H20, 6Rb, and [U-1 C]thiocyanate (S14CN, potassium salt). Biochemicals were from Boehringer (Mannheim, Germany); all other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany) or Sigma (St. Louis, Mo.). Determination of the cytoplasmic volume, proton gradient, and membrane potential. In the following experiments, cells were separated from the surrounding medium by silicone oil centrifugation (21). Experiments were carried out with 400-,ul Microfuge tubes filled with 30 ,ul of perchloric acid (20%) and 65 ,ul of silicone oil. If not mentioned otherwise, 100 ,ul of the cell suspension was added and centrifuged for 30 s. If necessary, cells were deenergized by addition of 100 ,uM carbonyl cyanide m-chlorophenylhydrazone (CCCP) or by glucose starvation. For measurement of the internal cell volume, we used 3H20 and [14C]taurine (29). Details were given elsewhere (10). We routinely defined a cell volume of 2.2 to 2.4 p./mg (dry mass). For measurement of the proton
Zymomonas mobilis is unique among bacteria in fermenting sugar anaerobically by the Entner-Doudoroff pathway and in catabolizing pyruvate to ethanol by pyruvate decarboxylase (9, 33). Although the carbohydrate metabolism of this organism has been studied in great detail (28, 34), knowledge about its transport processes is limited. Besides the well-documented highly active glucose uptake by facilitated diffusion (4, 14, 32), only transport of glutamine has recently been reported (2). It should be noted that, besides transport of glycerol in Escherichia coli (18) and glucose in Streptococcus bovis (30), transport of glucose in Z. mobilis is the only example of facilitated diffusion in procaryotes known so far. Because of the exclusive use of the Entner-Doudoroff pathway for ATP synthesis, energy supply is very limited in Z. mobilis. It is thus interesting to determine whether uptake systems other than the simple uniport system for glucose function according to well-known mechanisms of primary or secondary transport or whether facilitated diffusion is a general motive in Z. mobilis. After screening several amino acids for uptake activity in Z. mobilis, we chose glutamate for two reasons: first, it was the most active amino acid carrier system tested, and second, glutamate can be used by Z. mobilis as a nitrogen source. In different organisms, glutamate uptake is catalyzed by a large variety of systems. In E. coli, five uptake systems, both primary and secondary carriers (12, 17, 23, 31, 35), for anionic amino acids are known. Also, in gram-positive bacteria, various mechanisms have been observed, including secondary uptake coupled to H+ and Na+ (13) as well as ATP-dependent uptake (22, 27). In this report, we demonstrate that glutamate uptake in Z. mobilis is mediated by a secondary 2H+/glutamate symport system.
MATERIALS AND METHODS Organism and growth conditions. Z. mobilis subsp. mobilis ATCC 29191 was grown anaerobically overnight at 30°C in complex medium as described earlier (8). The medium *
Corresponding author. 7579
7580
RUHRMANN AND KRAMER
gradient, distribution of ["4C]benzoic acid (inside alkaline) and [14C]methylamine (inside acid) was used (29). Interference by active uptake of methylamine could be excluded. The results obtained were corrected for probe binding by using cells which were deenergized by glucose starvation or by addition of the protonophore CCCP or 0.01% cetylt-rimethylammonium bromide (CTAB). The membrane potential was determined by three different methods. First, distribution of the permeant cation [14Cjtetraphenylphosphonium bromide across the cytoplasmic membrane was monitored (20, 29). Second, we measured the distribution of "6Rb in the presence of up to 100 ,uM valinomycin (1, 29). Finally, we quantified the potential by determination of the distribution of [14C]KSCN (11) (see Results). Probe binding and uptake were not detected in this case. For determination of uptake of labelled glutamate, cells were preincubated at 30°C for 10 min in buffer before adding 100 to 200 ,uM labelled glutamate (450 to 900 Bq/nmol). In Km determination experiments, 0.01 to 5 mM glutamate was used. Aliquots (100 ,ul) of the cell suspension were taken after 30, 75, 120, and 180 s and separated by silicone oil centrifugation. Determination of glutamate by HPLC. Two hundred microliters of the cell suspension was separated by silicone oil centrifugation (see above), extracted in perchloric acid, and sonicated. The extracts were neutralized by adding 25 ,ul of a solution containing 5 M KOH-1M triethanolamine, kept on ice for at least 1 h to precipitate KCl04, and centrifuged. The supernatants were used for amino acid determination. Glutamate was detected fluorimetrically by reversed-phase highperformance liquid chromatography (HPLC) after precolumn derivatization with orthophthaldialdehyde reagent using an HP 1090 chromatograph. Details were given elsewhere (10). The total amount of glutamate was corrected by subtracting the fraction of external glutamate contaminating the sedimented cells. Determination of ATP concentration. The ATP content was detected by the firefly luciferin-luciferase assay. For calculation of the ATP concentration, internal and external standards had to be used. Details were given elsewhere (22). RESULTS Kinetics and basic properties of glutamate uptake. Uptake of labelled glutamate by Z. mobilis (Fig. 1) essentially depended on the presence of an energy source. When glucose was omitted or when glucose-supplemented cells were deenergized by the uncoupler CCCP, glutamate uptake was very low. Thus, in all further measurements of glutamate transport, glucose was present in saturating amounts. Uptake kinetics became nonlinear within the first few minutes of measurement. However, when glutamate transport was measured by monitoring the decrease in external glutamate by HPLC, linear uptake kinetics were obtained (data not shown). Both methods applied, uptake of labelled substrate and direct determination of external glutamate, led to essentially the same initial uptake rates. This shows that kinetic measurements by determination of both labelled and unlabelled internal substrate were affected by metabolism of glutamate within the cell. To circumvent this problem, we applied the transaminase reaction inhibitor aminooxyacetic acid (see also Fig. 4). For routine measurements, linear uptake kinetics up to 3 min could be obtained by adding a 3 mM concentration of this inhibitor, which obviously did not interfere with glutamate uptake (Fig. 1). Also in the presence of aminooxyacetic acid, the uptake kinetics became nonlinear when external glutamate concen-
J. BACTERIOL.
9=
20
0
L-
0 t
15
'a E
1
3
2
time (min) FIG. 1. Glutamate uptake at pH 5.5. Uptake of 200 ,uM labelled glutamate was measured after 10 min of preincubation with (A) and without (0) 150 mM glucose. Additionally, 3 mM aminooxyacetate (-) or 100 ,uM CCCP (*) was present.
trations above the Km were applied (not shown). This was however, caused not by interfering metabolism but by kinetic effects due to high internal glutamate accumulation (6, 16). By nonlinear regression of a Michaelis-Menten plot of initial uptake rates (not shown), a K_ of 1.05 +- 0.2 mM and a Vm. of 36.5 + 3 p,mol. min1 g (dry mass) of cells-1 at pH 5.5 were obtained. Since we measured uptake at relatively low pH values of S to 6, at which the growth conditions are optimal for Z. mobilis, it was necessary to define the true transport species. Glutamate has a pK2 value of.4.25; thus, both the glutamate anion (Glu-) and protonated glutamate (GluH) are possible substrates of the carrier protein. We measured both the Vm. and the apparent Km for glutamate uptake as functions of external pH (Table 1). The maximum uptake rate did not vary significantly within this pH range, whereas the apparent Km (calculated for total glutamate, i.e., the sum of Glu- and GluH) increased with increasing pH. When the apparent Km values were transferred into "true" values, assuming either Glu- or GluH as substrate, it became obvious that only unprotonated glutamate (Glu-) was accepted by the carrier. In the range of external pH between 5.0 and 7.0, the variations of the apparent Km which had to be assumed were TABLE 1. Kinetic constants for glutamate uptake at different
external pHs External pH
5.0 5.5 6.0 7.0
Km (mM)" Glutotal
0.75 1.05 1.06 1.60
± 0.21 ± 0.20 ± 0.21
± 0.50
Glu-
GluH
0.64 0.99 1.04 1.60
0.055 0.019 0.003
0.115
Vmi (nmg [dry mass] of cells-')
37.3 36.5 34.7 27.8
± 8.8 ± 3.0 ± 5.2
± 4.6
a The Kms for the anionic and undissociated forms of glutamate were calculated on the basis of a pK. of 4.25.
GLUTAMATE UPTAKE IN Z. MOBILIS
VOL. 174, 1992
2.5-fold for the anionic form and 38-fold for the protonated form. Glutamate and aspartate are taken up by the same transport system in Z. mobilis. Besides having very similar Kms for uptake, aspartate competitively inhibited glutamate uptake with an apparent Ki equal to the Km for glutamate uptake (data not shown). Addition of glutamine had no effect on glutamate uptake (not shown). We furthermore studied whether glutamate uptake is subject to induction or repression in Z. mobilis. In these experiments, either ammonium sulfate or glutamate was used as the nitrogen source in the growth medium. The carbon source had to be glucose in any case, since Z. mobilis is not able to use glutamate as a carbon source. We did not observe significant differences in the basic kinetic properties of glutamate uptake due to this variation. We thus conclude that glutamate uptake is constitutive in Z. mobilis. Determination of energetic parameters in Z. mobiis. When added to energized cells of Z. mobilis, glutamate is significantly accumulated within the cytosol. To investigate the possible driving forces for this uptake system, we quantitated the membrane potential (A*i), pH gradient (ApH), and cytosolic ATP. For determination of internal ATP concentration and of ApH, we used conventional procedures, as described in Materials and Methods. However, determination of Aj proved to be very difficult with Z. mobilis. As reported earlier (19), the lipophilic probe tetraphenylphosphonium bromide did not distribute across the membrane according to the electrical gradient. The strong adsorption of the probe was not significantly changed by deenergization with 100 ,uM CCCP. This phenomenon is well known from other gram-negative bacteria, such as E. coli (3). In these cases, however, the problems can to some extent be overcome by treatment with EDTA (25). In Z. mobilis, various experimental protocols using EDTA for destabilization of the outer membrane did not lead to correct distribution of the lipophilic probe. Also, addition of tetraphenylboron for saturation of unspecific binding sites had no positive effect. The use of 86Rb in the presence of valinomycin, which in general is a very useful method for determination of the membrane potential (1, 20, 29), was also not applicable, because valinomycin did not insert into the plasma membrane of Z. mobilis. Addition of EDTA did not improve these results. The only probe which proved to sense the membrane potential correctly was S14CN-, which, however, in principle is not a well-suited probe under these conditions. Because of its negative charge, the cytosolic SCN- concentration is decreased according to the Nernst potential with increasing electrical potentials (positive outside). This limits the range of measurement to membrane potentials not lower (more negative) than about -100 mV. As known from bacteria in general (5, 6, 26), the cells tend to keep the proton motive force constant when changing the external pH, i.e., varying the contribution of ZApH according to the equation Ap = A* - ZApH. In fact, when measuring both ApH ([ 4C]benzoate) and A* (S14CN-) at low external pH, we found that between pH 4 and 5, where both components of the proton motive force could be determined directly, Ap stayed relatively constant at about -132 to -138 mV, although both components, ApH and A+, varied significantly (Fig. 2). Between external pHs of 4 and 6.5 the cells showed effective pH homeostasis, maintaining an internal pH of about 6.0. Whereas up to pH 5.5, Ap could be calculated directly, above pH 5.5, the electrical part of the chemiosmotic potential could be calculated only by assuming a constant Ap of -137 mV (see above) together
7581
-120
0
E N
-60-
60
4
5
6
7
external pH FIG. 2. Dependence of ApH (T), A* (0), and Ap (E) on external pH. The pH gradient (from pH 4 to 7.5) and Aji (from pH 4 to 5.5) were measured as described in the text. The standard errors given for A* and ApH measurements are based on 6 individual determinations; for membrane potential at pH 5 and 5.5, 10 determinations each were carried out. The membrane potential for pHs above 5.5 was calculated assuming a constant Ap of -137 mV in this range (dashed and dotted lines) (see text).
with the measured value for ApH (Fig. 2). A further validation for this extrapolation is provided by the data shown in Table 3, as discussed below. Ener coupling of glutamate uptake. (i) characterization as a secondary system. As shown above (Fig. 1), glutamate uptake could be observed only in the presence of an appropriate energy source and was inhibited by addition of the uncoupler CCCP. When the energetic parameters in this experiment were determined (Fig. 1), it turned out that after addition of glucose at pH 5.5, we observed not only an increase in ApH (from 0 to 0.6 pH units) and A4 (from less than -10 to about -100 mV) but also an increase in internal ATP from below 0.1 mM (the detection limit) to 4 mM. Thus, the possible contributions of the different energetic parameters to the observed glutamate uptake had to be distinguished. Unfortunately, the common reagents used for these purposes were not applicable with Z. mobilis. Not only the ionophore valinomycin, as mentioned above, but also nigericin and dicyclohexylcarbodiimide, an inhibitor of the F1FoATPase, were not effective (data not shown). The only compound which we could use successfully was the uncoupler CCCP in high concentrations (Fig. 3). When a CCCP concentration greater than 50 ,uM was applied, the membrane potential was reduced significantly, in contrast to the cytosolic ATP concentration, which stayed constant when up to 100 ,uM CCCP was added. Simultaneously with added uncoupler, the glutamate uptake rate was decreased to comparable extent. The experiments whose results are shown in Fig. 3 were carried out at an external pH of 5.5. We obtained essentially the same results when measuring at pH 6.5, at which an inverse ApH (inside acid) is present (not shown). Also in this case, a more or less perfect correlation to A, was observed. The same correlation between A* and glutamate uptake could be obtained by simply using high external concentrations of NaCl or KCI. At salt concentrations above 150 mM, both membrane potential and glutamate uptake rate decreased significantly, whereas internal ATP stayed constant (data not shown).
J. BACrERIOL.
RUHRMANN AND KRAMER
7582
3000 1
744 \ ->-120 T
=
-
2f 80 0.31 E E1 E -80
T
T
(U
X
_0
'
0
cn
_
0
20-60 4~T
6
8
l
2
1000*
i
300
0
10
0
E -20-
E
0
A
._
-
80
-
70 *
_x -60 (3
t
8
6
A9 50
-
A ...'
\
q
4 C h0
A
I
-W
._
30-
'q
E 8
E 100-
EE cm10
10 1
-40 Al' 3 -30
..,&
le..S-
A>
JOURNAL OF BACrERIOLOGY, Dec. 1992, p. 7579-7584
0021-9193/92/237579-06$02.00/0 Copyright e 1992, American Society for Microbiology
Mechanism of Glutamate Uptake in Zymomonas mobilis JUTrA RUHRMANN AND REINHARD KRAMER* Institut fiir Biotechnologie I, Forschungszentrum Julich, Postfach 1913, 5170 Julich, Germany Received 17 August 1992/Accepted 4 October 1992
The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanolproducing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of3.5 to 4 mM. Because ofeffective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed ofa pH gradient of0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high V. of 40 ,umol min1 g (dry mass) of celis- and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.
contained (per liter) 50 g of glucose, 10 g of Bacto yeast extract, 1 g of KH2PO4, 1 g of (NH4)2SO4, and 0.5 g of MgSO4. 7H20 (pH 5.0). For uptake experiments, cells were harvested at a cell density of 0.35 to 0.5 mg (dry mass) per ml. The cells were washed twice in buffer containing 100 mM MES (morpholineethanesulfonic acid), 10 mM NaCl, 10 mM KCI, and 3 to 5% glucose and suspended in the same buffer at a cell density of 2.1 to 2.4 mg (dry mass) per ml. If not mentioned otherwise, 3 mM aminooxyacetic acid, an inhibitor of aminotransferases, was added. For investigating the inducibility of the glutamate uptake system, cells were grown in mineral medium, modified from that described in reference 16, which contained (per liter) 1.0 g of MgSO4- 7H20, 3.48 g of KH2PO4, 0.2 g of citric acid monohydrate, 0.01 g of FeSO4. 7H20, 19.52 g of MES, and 4.4 g of glutamate (30 mM) or 1.98 g of (NH4)2SO4 as an N source (pH 5.8). After sterilization, calcium pantothenate and biotin were added to a final concentrations of 1.5 mg of each per liter. Chemicals. Radiochemicals were purchased from Amersham International (Amersham, Buckinghamshire, United Kinlgdom). The following labelled compounds were used: [U-'4C]glutamic acid, [U-14C]tetraphenylphosphonium bromide, [U-14Ctaurine, 2-[1494methylamine, [U-14C]benzoic acid, 3H20, 6Rb, and [U-1 C]thiocyanate (S14CN, potassium salt). Biochemicals were from Boehringer (Mannheim, Germany); all other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany) or Sigma (St. Louis, Mo.). Determination of the cytoplasmic volume, proton gradient, and membrane potential. In the following experiments, cells were separated from the surrounding medium by silicone oil centrifugation (21). Experiments were carried out with 400-,ul Microfuge tubes filled with 30 ,ul of perchloric acid (20%) and 65 ,ul of silicone oil. If not mentioned otherwise, 100 ,ul of the cell suspension was added and centrifuged for 30 s. If necessary, cells were deenergized by addition of 100 ,uM carbonyl cyanide m-chlorophenylhydrazone (CCCP) or by glucose starvation. For measurement of the internal cell volume, we used 3H20 and [14C]taurine (29). Details were given elsewhere (10). We routinely defined a cell volume of 2.2 to 2.4 p./mg (dry mass). For measurement of the proton
Zymomonas mobilis is unique among bacteria in fermenting sugar anaerobically by the Entner-Doudoroff pathway and in catabolizing pyruvate to ethanol by pyruvate decarboxylase (9, 33). Although the carbohydrate metabolism of this organism has been studied in great detail (28, 34), knowledge about its transport processes is limited. Besides the well-documented highly active glucose uptake by facilitated diffusion (4, 14, 32), only transport of glutamine has recently been reported (2). It should be noted that, besides transport of glycerol in Escherichia coli (18) and glucose in Streptococcus bovis (30), transport of glucose in Z. mobilis is the only example of facilitated diffusion in procaryotes known so far. Because of the exclusive use of the Entner-Doudoroff pathway for ATP synthesis, energy supply is very limited in Z. mobilis. It is thus interesting to determine whether uptake systems other than the simple uniport system for glucose function according to well-known mechanisms of primary or secondary transport or whether facilitated diffusion is a general motive in Z. mobilis. After screening several amino acids for uptake activity in Z. mobilis, we chose glutamate for two reasons: first, it was the most active amino acid carrier system tested, and second, glutamate can be used by Z. mobilis as a nitrogen source. In different organisms, glutamate uptake is catalyzed by a large variety of systems. In E. coli, five uptake systems, both primary and secondary carriers (12, 17, 23, 31, 35), for anionic amino acids are known. Also, in gram-positive bacteria, various mechanisms have been observed, including secondary uptake coupled to H+ and Na+ (13) as well as ATP-dependent uptake (22, 27). In this report, we demonstrate that glutamate uptake in Z. mobilis is mediated by a secondary 2H+/glutamate symport system.
MATERIALS AND METHODS Organism and growth conditions. Z. mobilis subsp. mobilis ATCC 29191 was grown anaerobically overnight at 30°C in complex medium as described earlier (8). The medium *
Corresponding author. 7579
7580
RUHRMANN AND KRAMER
gradient, distribution of ["4C]benzoic acid (inside alkaline) and [14C]methylamine (inside acid) was used (29). Interference by active uptake of methylamine could be excluded. The results obtained were corrected for probe binding by using cells which were deenergized by glucose starvation or by addition of the protonophore CCCP or 0.01% cetylt-rimethylammonium bromide (CTAB). The membrane potential was determined by three different methods. First, distribution of the permeant cation [14Cjtetraphenylphosphonium bromide across the cytoplasmic membrane was monitored (20, 29). Second, we measured the distribution of "6Rb in the presence of up to 100 ,uM valinomycin (1, 29). Finally, we quantified the potential by determination of the distribution of [14C]KSCN (11) (see Results). Probe binding and uptake were not detected in this case. For determination of uptake of labelled glutamate, cells were preincubated at 30°C for 10 min in buffer before adding 100 to 200 ,uM labelled glutamate (450 to 900 Bq/nmol). In Km determination experiments, 0.01 to 5 mM glutamate was used. Aliquots (100 ,ul) of the cell suspension were taken after 30, 75, 120, and 180 s and separated by silicone oil centrifugation. Determination of glutamate by HPLC. Two hundred microliters of the cell suspension was separated by silicone oil centrifugation (see above), extracted in perchloric acid, and sonicated. The extracts were neutralized by adding 25 ,ul of a solution containing 5 M KOH-1M triethanolamine, kept on ice for at least 1 h to precipitate KCl04, and centrifuged. The supernatants were used for amino acid determination. Glutamate was detected fluorimetrically by reversed-phase highperformance liquid chromatography (HPLC) after precolumn derivatization with orthophthaldialdehyde reagent using an HP 1090 chromatograph. Details were given elsewhere (10). The total amount of glutamate was corrected by subtracting the fraction of external glutamate contaminating the sedimented cells. Determination of ATP concentration. The ATP content was detected by the firefly luciferin-luciferase assay. For calculation of the ATP concentration, internal and external standards had to be used. Details were given elsewhere (22). RESULTS Kinetics and basic properties of glutamate uptake. Uptake of labelled glutamate by Z. mobilis (Fig. 1) essentially depended on the presence of an energy source. When glucose was omitted or when glucose-supplemented cells were deenergized by the uncoupler CCCP, glutamate uptake was very low. Thus, in all further measurements of glutamate transport, glucose was present in saturating amounts. Uptake kinetics became nonlinear within the first few minutes of measurement. However, when glutamate transport was measured by monitoring the decrease in external glutamate by HPLC, linear uptake kinetics were obtained (data not shown). Both methods applied, uptake of labelled substrate and direct determination of external glutamate, led to essentially the same initial uptake rates. This shows that kinetic measurements by determination of both labelled and unlabelled internal substrate were affected by metabolism of glutamate within the cell. To circumvent this problem, we applied the transaminase reaction inhibitor aminooxyacetic acid (see also Fig. 4). For routine measurements, linear uptake kinetics up to 3 min could be obtained by adding a 3 mM concentration of this inhibitor, which obviously did not interfere with glutamate uptake (Fig. 1). Also in the presence of aminooxyacetic acid, the uptake kinetics became nonlinear when external glutamate concen-
J. BACTERIOL.
9=
20
0
L-
0 t
15
'a E
1
3
2
time (min) FIG. 1. Glutamate uptake at pH 5.5. Uptake of 200 ,uM labelled glutamate was measured after 10 min of preincubation with (A) and without (0) 150 mM glucose. Additionally, 3 mM aminooxyacetate (-) or 100 ,uM CCCP (*) was present.
trations above the Km were applied (not shown). This was however, caused not by interfering metabolism but by kinetic effects due to high internal glutamate accumulation (6, 16). By nonlinear regression of a Michaelis-Menten plot of initial uptake rates (not shown), a K_ of 1.05 +- 0.2 mM and a Vm. of 36.5 + 3 p,mol. min1 g (dry mass) of cells-1 at pH 5.5 were obtained. Since we measured uptake at relatively low pH values of S to 6, at which the growth conditions are optimal for Z. mobilis, it was necessary to define the true transport species. Glutamate has a pK2 value of.4.25; thus, both the glutamate anion (Glu-) and protonated glutamate (GluH) are possible substrates of the carrier protein. We measured both the Vm. and the apparent Km for glutamate uptake as functions of external pH (Table 1). The maximum uptake rate did not vary significantly within this pH range, whereas the apparent Km (calculated for total glutamate, i.e., the sum of Glu- and GluH) increased with increasing pH. When the apparent Km values were transferred into "true" values, assuming either Glu- or GluH as substrate, it became obvious that only unprotonated glutamate (Glu-) was accepted by the carrier. In the range of external pH between 5.0 and 7.0, the variations of the apparent Km which had to be assumed were TABLE 1. Kinetic constants for glutamate uptake at different
external pHs External pH
5.0 5.5 6.0 7.0
Km (mM)" Glutotal
0.75 1.05 1.06 1.60
± 0.21 ± 0.20 ± 0.21
± 0.50
Glu-
GluH
0.64 0.99 1.04 1.60
0.055 0.019 0.003
0.115
Vmi (nmg [dry mass] of cells-')
37.3 36.5 34.7 27.8
± 8.8 ± 3.0 ± 5.2
± 4.6
a The Kms for the anionic and undissociated forms of glutamate were calculated on the basis of a pK. of 4.25.
GLUTAMATE UPTAKE IN Z. MOBILIS
VOL. 174, 1992
2.5-fold for the anionic form and 38-fold for the protonated form. Glutamate and aspartate are taken up by the same transport system in Z. mobilis. Besides having very similar Kms for uptake, aspartate competitively inhibited glutamate uptake with an apparent Ki equal to the Km for glutamate uptake (data not shown). Addition of glutamine had no effect on glutamate uptake (not shown). We furthermore studied whether glutamate uptake is subject to induction or repression in Z. mobilis. In these experiments, either ammonium sulfate or glutamate was used as the nitrogen source in the growth medium. The carbon source had to be glucose in any case, since Z. mobilis is not able to use glutamate as a carbon source. We did not observe significant differences in the basic kinetic properties of glutamate uptake due to this variation. We thus conclude that glutamate uptake is constitutive in Z. mobilis. Determination of energetic parameters in Z. mobiis. When added to energized cells of Z. mobilis, glutamate is significantly accumulated within the cytosol. To investigate the possible driving forces for this uptake system, we quantitated the membrane potential (A*i), pH gradient (ApH), and cytosolic ATP. For determination of internal ATP concentration and of ApH, we used conventional procedures, as described in Materials and Methods. However, determination of Aj proved to be very difficult with Z. mobilis. As reported earlier (19), the lipophilic probe tetraphenylphosphonium bromide did not distribute across the membrane according to the electrical gradient. The strong adsorption of the probe was not significantly changed by deenergization with 100 ,uM CCCP. This phenomenon is well known from other gram-negative bacteria, such as E. coli (3). In these cases, however, the problems can to some extent be overcome by treatment with EDTA (25). In Z. mobilis, various experimental protocols using EDTA for destabilization of the outer membrane did not lead to correct distribution of the lipophilic probe. Also, addition of tetraphenylboron for saturation of unspecific binding sites had no positive effect. The use of 86Rb in the presence of valinomycin, which in general is a very useful method for determination of the membrane potential (1, 20, 29), was also not applicable, because valinomycin did not insert into the plasma membrane of Z. mobilis. Addition of EDTA did not improve these results. The only probe which proved to sense the membrane potential correctly was S14CN-, which, however, in principle is not a well-suited probe under these conditions. Because of its negative charge, the cytosolic SCN- concentration is decreased according to the Nernst potential with increasing electrical potentials (positive outside). This limits the range of measurement to membrane potentials not lower (more negative) than about -100 mV. As known from bacteria in general (5, 6, 26), the cells tend to keep the proton motive force constant when changing the external pH, i.e., varying the contribution of ZApH according to the equation Ap = A* - ZApH. In fact, when measuring both ApH ([ 4C]benzoate) and A* (S14CN-) at low external pH, we found that between pH 4 and 5, where both components of the proton motive force could be determined directly, Ap stayed relatively constant at about -132 to -138 mV, although both components, ApH and A+, varied significantly (Fig. 2). Between external pHs of 4 and 6.5 the cells showed effective pH homeostasis, maintaining an internal pH of about 6.0. Whereas up to pH 5.5, Ap could be calculated directly, above pH 5.5, the electrical part of the chemiosmotic potential could be calculated only by assuming a constant Ap of -137 mV (see above) together
7581
-120
0
E N
-60-
60
4
5
6
7
external pH FIG. 2. Dependence of ApH (T), A* (0), and Ap (E) on external pH. The pH gradient (from pH 4 to 7.5) and Aji (from pH 4 to 5.5) were measured as described in the text. The standard errors given for A* and ApH measurements are based on 6 individual determinations; for membrane potential at pH 5 and 5.5, 10 determinations each were carried out. The membrane potential for pHs above 5.5 was calculated assuming a constant Ap of -137 mV in this range (dashed and dotted lines) (see text).
with the measured value for ApH (Fig. 2). A further validation for this extrapolation is provided by the data shown in Table 3, as discussed below. Ener coupling of glutamate uptake. (i) characterization as a secondary system. As shown above (Fig. 1), glutamate uptake could be observed only in the presence of an appropriate energy source and was inhibited by addition of the uncoupler CCCP. When the energetic parameters in this experiment were determined (Fig. 1), it turned out that after addition of glucose at pH 5.5, we observed not only an increase in ApH (from 0 to 0.6 pH units) and A4 (from less than -10 to about -100 mV) but also an increase in internal ATP from below 0.1 mM (the detection limit) to 4 mM. Thus, the possible contributions of the different energetic parameters to the observed glutamate uptake had to be distinguished. Unfortunately, the common reagents used for these purposes were not applicable with Z. mobilis. Not only the ionophore valinomycin, as mentioned above, but also nigericin and dicyclohexylcarbodiimide, an inhibitor of the F1FoATPase, were not effective (data not shown). The only compound which we could use successfully was the uncoupler CCCP in high concentrations (Fig. 3). When a CCCP concentration greater than 50 ,uM was applied, the membrane potential was reduced significantly, in contrast to the cytosolic ATP concentration, which stayed constant when up to 100 ,uM CCCP was added. Simultaneously with added uncoupler, the glutamate uptake rate was decreased to comparable extent. The experiments whose results are shown in Fig. 3 were carried out at an external pH of 5.5. We obtained essentially the same results when measuring at pH 6.5, at which an inverse ApH (inside acid) is present (not shown). Also in this case, a more or less perfect correlation to A, was observed. The same correlation between A* and glutamate uptake could be obtained by simply using high external concentrations of NaCl or KCI. At salt concentrations above 150 mM, both membrane potential and glutamate uptake rate decreased significantly, whereas internal ATP stayed constant (data not shown).
J. BACrERIOL.
RUHRMANN AND KRAMER
7582
3000 1
744 \ ->-120 T
=
-
2f 80 0.31 E E1 E -80
T
T
(U
X
_0
'
0
cn
_
0
20-60 4~T
6
8
l
2
1000*
i
300
0
10
0
E -20-
E
0
A
._
-
80
-
70 *
_x -60 (3
t
8
6
A9 50
-
A ...'
\
q
4 C h0
A
I
-W
._
30-
'q
E 8
E 100-
EE cm10
10 1
-40 Al' 3 -30
..,&
le..S-
A>
