Vol. 127, No. 3 Printed in U.S.A.
JouNAiL oF BACTZRIoLOGY, Sept. 1976, p. 1265-1264 Copyright C 1976 American Society for Microbiology
Energy Coupling in the Active Transport of Proline and Glutamate by the Photosynthetic Halophile Ectothiorhodospira halophila CLIFFORD A. RINEHART AND JERRY S. HUBBARD* School ofBiology, Georgia Institute of Technology, Atlanta, Georgia 30332 Received for publication 5 April 1976
When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by darkexposed cells. CCCP strongly inhibited glutamate uptake in the dark. The lightdependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.
Gram-negative organisms have been shown
membrane-bound systems are not yet completely understood. Initially, Kaback and Barnes (14) proposed a working model based on oxidation-reduction coupling at discrete portions of the respiratory chain. Others (2, 7, 11, 16, 21) interpreted their results as supporting Mitchell's chemiosmotic theory of membrane transport (22, 23). This theory states that the energy-transducing reactions of electron transport, ATP synthesis, and active transport are phosphatase (ATPase). The second class is linked energetically and mechanistically by the termed a membrane-bound or respiratory- proton-motive force. The force is derived from linked system. These shock-resistant systems the efflux of protons during electron transport contain permeases but do not contain dissocia- and/or by hydrolysis of ATP by the membrane ble binding proteins. The driving force for the ATPase (2, 4). The proton-motive force (Ap) is second type is not necessarily derived from composed of transmembrane gradients of elecATP. Systems of the latter class have been trical and chemical potentials: Ap = A* extensively studied in membrane vesicles (see ZApH, where A* is the membrane potential (interior negative), ApH is the pH gradient reference 13 for a review). The mechanisms of energy coupling to the (interior alkaline), and Z is a factor to convert
to possess two distinct classes of active transport systems that do not chemically modify the substrate (3, 4, 31). Shock-sensitive systems contain a dissociable binding protein in addition to the permease. These shockable systems are coupled to the phosphate bond energy of adenosine triphosphate (ATP) or a similar high-energy compound by an unknown mechanism not using the membrane adenosine tri-
1255
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RINEHART AND HUBBARD
pH units into electrical units, equaling about 59 mV/pH unit at 250C. These separate components of the proton-motive force have been studied individually by the use of specific ionophores and inhibitors. From these studies Hamilton and Niven (6) formulated a model to explain the driving force for the uptake of various amino acids by Staphylococcus aureus. Basic amino acid uptake was driven by Atp via a uniport mechanism, neutral amino acid uptake was driven by Ap via an H+ symport mechanism, and acidic amino acid uptake was driven by -ZApH via an H+ symport mechanism. Only preliminary characterizations have been made on active transport systems of photosynthetic bacteria. In studies with Chromatium sp. strain D, it was concluded that this organism lacks systems of sufficiently high affinity for efficient amino acid transport (30). The uptake of dicarboxylic acids and pyruvate have been studied in the purple, nonsulfur bacterium Rhodopseudomonas spheroides (5). Hellingwerfet al. (9) recently demonstrated an alanine transport system in cells and membrane vesicles of R. spheroides. The energy to drive alanine uptake could be derived from aerobic respiration in the dark or from anaerobic, photosynthetic electron transport. To our knowledge active transport has not been demonstrated in obligately photosynthetic bacteria. Ectothiorhodospira halophila was originally isolated from a salt pond in southern Oregon by Raymond and Sistrom (27, 28). This purple, sulfur bacterium is obligately anaerobic and photosynthetic. E. halophila is also obligately halophilic, requiring at least 9% NaCl for growth. The species grows well between 11 and 22% NaCl and will grow slowly at 30% NaCl. The photosynthetic apparatus in E. halophila is contained in a complex system of lamellar membranes (27). The objectives of the present study were to characterize the proline and glutamate transport systems in E. halophila and to define the mechanism(s) by which energy is coupled to these processes. MATERIALS AND METHODS Cell suspensions. The culture ofE. halophila was generously provided by W. R. Sistrom. The anaerobic, photosynthetic growth conditions were similar to those previously described (27). A single exception was that our medium contained 0.2% sodium acetate instead of succinate. Thiosulfate was used as the source of reduced sulfur. A 20% NaCl level was used throughout. Cultures were grown in 168-ml milk dilution bottles which were completely filled. A stock culture was stored at 4°C. Cultures were incubated at 41°C under incandescent light for 2 days.
J. BACTERIOL.
Cultures were harvested by centrifugation, washed two times in a buffer of 1.0 M NaCl and 0.02 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8.0, and resuspended in the same buffer to a turbidity of 150 Klett units in a Klett-Summerson colorimeter equipped with a no. 66 filter. This turbidity corresponds to 0.41 mg of cell protein per ml. All harvesting and washing operations were performed at 40C. The cells were kept on ice under ordinary lab light prior to the transport assays. The cells retained approximately 50% of their activity for the uptake of proline and glutamate 24 h after harvesting. Amino acid uptake. Cell suspensions were incubated in glass tubes (16 by 100 mm) at 41.5°C in a Braun Warburg apparatus equipped with incandescent lamps to the exterior of the plexiglass bath. The light intensity reaching the tubes was about 2 mW/ cm2 as measured with a Kettering YSI model 65 radiometer. Dark incubations were conducted in aluminum foil-covered tubes under laboratory lights reduced as far as practical. Unless otherwise indicated, all incubations were conducted with N2 bubbling through the medium at about 80 cm3/min. After a preincubation of 5 min 14C-labeled amino acid was added to the cell suspension and 0.5-ml portions were removed at intervals. The cells were rapidly collected on membrane filters (Gelman GA6, 0.47 jAm, 25-mm diameter) and washed with 1 ml of chilled Tris-1 M NaCl buffer. The filter disks were then placed in scintillation vials and dissolved in a solution containing 1 ml of water, 2 ml of methyl cellosolve, and 10 ml of a cocktail made of dioxane, 10% naphthalene (wt/vol), and 0.5% diphenyloxazole (wt/vol). Samples were counted on a Beckmann LS100C liquid scintillation counter. Unless otherwise indicated, the "4C-labeled amino acids were diluted in 0.1 mM 12C-labeled amino acid and added at levels to give a final concentration of 2 ,uM in the incubation mixture. Initial rates were determined over the 4-min interval immediately after "4C-labeled amino acid addition. All measurements were corrected for retention of "4C-labeled amino acid by the filters in zero time controls. The retention values were 0.09 nmol for [14C]glutamate and 0.05 nmol for
['4C]proline. Uptake of the [3H]triphenylmethylphosphonium cation ([3H]TPMP+) was assayed in a similar manner as described for the amino acids. The membrane filters retained 0.8 nmol of TPMP+ in zero-time controls. Membrane vesicles. Cells suspended in 0.02 M Tris buffer containing 20% sucrose and 0.01 M ethylenediaminetetracetic acid were treated with muramidase (0.5 mg/ml) for 20 min at 23°C. The resulting spheroplasts were then centrifuged and resuspended in the Tris-1 M NaCl buffer to which was added 10 mM sodium ascorbate and 20 ,uM dichloroindophenol to maintain the recommended redox potential (9). Membrane vesicles were then prepared by sonic treatment of the spheroplasts for four 30-s intervals with a Heat Systems Co. Sonifier Cell Disrupter equipped with a microtip. The preparation was incubated with deoxyribonuclease (0.1 mg/ ml) for 15 min to reduce the viscosity and then
VOL. 127, 1976
AMINO ACID TRANSPORT BY E. HALOPHILA
centrifuged for 20 min at 5,000 x g to remove any remaining whole cells. The vesicles were then harvested by centrifugation for 20 min at 45,000 x g and resuspended using a syringe equipped with a 21gauge needle. The preparation was examined microscopically to ascertain that no whole cells remained. In some experiments the intravesicular salt composition was varied by sonically treating the spheroplasts in buffer containing NaCl-KCl mixtures. All preparative operations were at 4 to 5°C. Analytical. Thin-layer chromatography of amino acids was done on silica gel plates (60F-254) obtained from EM Laboratories. The solvent systems were sec-butyl alcohol-formic acid-water (7:1:2) and chloroform-methanol-17% NH4OH solution (4:4:2). Autoradiography was accomplished by exposing du Pont Cronex medical X-ray film to the thin-layer plates. Enzymatic determination of glutamic acid was made by using glutamate decarboxylase prepared from dried cells of Escherichia coli Crookes strain (ATCC 8739). Protein was determined on portions of cells lysed in 0.02 M Tris buffer, using the method of Lowry et al. (20) with bovine serum albumin as the standard. Intracellular ATP was extracted by injecting 0.1 ml of cell suspension into 0.9 ml of boiling 0.02 M Tris, pH 7.4, and the boiling was continued for an additional 2 min. The ATP content of the extract was determined by using the firefly assay as previously reported (11). Chemicals. i_[U-14C]proline (specific activity, 232 Ci/mol) and L_[U-14C]glutamic acid (specific activity, 290 Ci/mol) were obtained from New England Nuclear Corp. [1-14C]glutamic acid (specific activity, 4.84 Ci/mol) was from Calbiochem. Tritiated triphenylmethylphosphonium bromide (specific activity, 114 Ci/mol) was a gift from H. R. Kaback. Unlabeled triphenylmethylphosphonium bromide (K & K Laboratories) was used in a final concentration of 1 mM as an inhibitor and at 0.02 mM as a carrier for the labeled TPMP+. N,N'-dicyclohexylcarbodiimide (DCCD) was from Eastman and was used in a 0.1 mM final concentration. Carbonylcyanide-m-chlorophenylhydrazone (CCCP), used at a 0.01 mM final concentration, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), used at a 0.1 mM final concentration, and valinomycin, used at a 2-,g/ml final concentration, were all from Sigma. The purified and stabilized luciferin-luciferase mixture was from du Pont. All other chemicals were reagent grade and were obtained from standard sources.
RESULTS General aspects of glutamate and proline uptake. Although E. halophila will not grow in media containing less than 1.6 M NaCl, the maximum rate of glutamate uptake was effected by cells suspended in 1 M NaCl (data not shown). The initial rate of ['4C]proline uptake was highest with cells in 1 to 2 M NaCl (data not shown). Thus, 1 M NaCl was chosen as the salt concentration for both glutamate and proline assays so as to achieve highest activities and to simplify preparative procedures.
1257
Several experiments were performed to ascertain that the amino acids were actually transported against a concentration gradient and not chemically altered by the cells. Analyses were made on 14C-labeled amino acids that had been taken up by the cells and then recovered by lysing the cells with distilled water. In the case of [14C]proline the lysate was evaporated and then extracted with absolute ethanol. Over 95% of the total 14C was recovered in this ethanol-soluble fraction. Thin-layer chromatographic and autoradiographic analysis showed that over 90% of the ethanol-soluble 14C was contained in one spot which exhibited chromatographic behavior identical to authentic proline. For the analysis of glutamic acid the cells were allowed to take up [1-14C]glutamate and then lysed as above. Essentially all of the 14C was recovered in the watersoluble fraction. This 14C-labeled extract was mixed with _[12C]glutamic acid and then treated with glutamate decarboxylase, an enzyme which specifically cleaves the number one carbon of i-glutamate. The CO2 evolved was trapped in hyamine hydroxide and its radioactivity was determined. The finding that the "4Clabeled fraction recovered from the cells was decarboxylated to the same extent as authentic [1-_4C]glutamic acid was evidence that the accumulated amino acid had not been chemically altered. From rough calculations it is apparent that the uptake of both glutamate and proline are concentrative. For example, in typical experiments about half (1 nmol) of the available amino acid (2 nmol/ml) is accumulated. Assuming that the intracellular volume is 80% of the total cell volume of 4.1 ,ul/ml, then 1 nmol of amino acid is contained in 3.3 ,ul of the intracellular space. Thus, the concentration of glutamate or proline is about 300 times greater inside the cells than outside. The uptake of both proline and glutamate were inhibited by bubbling 02 through the incubation mixture or by increasing the light intensity to 30 mW/cm2 (data not shown). As will be shown below, E. halophila cells take up glutamate in the dark. Several lines of evidence contraindicate the possibility that this activity reflects some unknown form of dark respiration whereby electrons are transferred to an organic or inorganic acceptor. The uptake of glutamate in the dark was not stimulated by providing oxygen; in fact, it was inhibited. Also, neither thiosulfate nor acetate stimulated the dark activity under anaerobic conditions. This activity was not enhanced by providing the electron donors ascorbate plus phenazine methosulfate or ascorbate plus N,N,N',N'-tet-
1258
J. BACTERIOL.
RINEHART AND HUBBARD
ramethyl-1,4-phenyldiamine (TMPD). The use of phenazine methosulfate, TMPD, or methyl viologen as potential electron acceptors did not stimulate this dark activity. Evidence is presented below that this dark process is driven by the hydrolysis of ATP by membrane ATPase. We also excluded the possibility that dissociable binding proteins were involved in the proline or glutamate transport systems. Subjecting cells or spheroplasts to rapid changes in osmotic strength (1 to 3.4 M NaCl) did not inactivate either system. Also, the observation of active systems in membrane vesicle preparations (see Fig. 8) argues against dissociable components being parts of the transport systems. Proline transport system. Proline uptake by illuminated cells proceeds until the maximum intracellular concentration is approached at about 30 min (Fig. 1). The uptake does not occur in the dark, nor is the uptake stimulated by preincubating the cells in the light, placing them in the dark, and immediately adding [14C]proline. An efflux of [14C]proline occurs when an excess of [12C]proline is added to the incubation after the cells have previously accumulated ['4C]proline. A loss of ['4C]proline from the cells also occurs when the light is turned off (Fig. 1). Photophosphorylation and proline uptake. The intracellular ATP reaches a maximum level of about 3.5 nmol/mg of cell protein in less than 5 min of incubation in the light (Fig. 2). This level is maintained during the course of proline uptake in the light. A slow drop in the intracellular ATP level occurs when illumination is discontinued. Although the cells initially contained as much ATP in the dark (after preillumination) as in irradiated cells, this amount was not associated with proline uptake. Inhibition of proline uptake. CCCP, a compound which renders membranes permeable to protons (24) and uncouples photophosphorylation (12), almost completely inhibits proline uptake (Fig. 3). HQNO, an inhibitor of photosynthetic electron transport (12), is also a strong inhibitor. The ATPase inhibitor (8) DCCD stimulates proline accumulation. In Fig. 4 the time course of proline uptake and ATP levels were compared in the presence of DCCD or CCCP. The addition of DCCD causes a drop in the intracellular ATP level, while the uptake of [14C]proline proceeds rapidly. In contrast, with CCCP present ATP was maintained at the initial level and proline uptake was almost abolished. The maintenance of an intermediate level of ATP in cells exposed to CCCP suggests that some photophosphoryla-
4.0
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TIME, min FIG. 1. Proline uptake and efflux in the light and dark. Symbols: 0, standard conditions in light; O, incubated in light for 10 min and then made dark, and the incubation was continued; 0, incubated in light for 10 min, 2 mM ["2C]proline was then added, and the light incubation was continued; A, dark throughout; A, after 5 min of preincubation in the light, the tubes were placed in the dark concomitant with the addition of ['4C]proline at zero time. ATP F 14C-pro I level LSuptakeE
.92.0_
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-)
-
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4.0E E
dk
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-5
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TIME (mh)
FIG. 2. Time course of proline uptake and intracellular ATP levels. hp = cells illuminated throughout; hi, -- dK = cells were preilluminated for 5 min and then placed in the dark. ['4C]proline was added at zero time in both experiments.
tion was occurring. If CCCP had completely blocked photophosphorylation, the consuming reactions of the cell should deplete the ATP as was observed with DCCDYpresent (Fig. 4). We cannot exclude the possibility that the ATP formation in the presence of CCCP was occurring by substrate level phosphorylation, but
AMINO ACID TRANSPORT BY E. HALOPHILA
VOL. 127, 1976
1259
line). Thus, the inhibition of proline uptake by TPMP+ suggests that Aq, is an essential component of the driving force. From the findings of Fig. 2 and 4 it is apparent that proline uptake is not directly related to the ATP level of the cells. Moreover, the results with DCCD contraindicated ATP hydrolysis as being of importance in providing the driving force. In addition to its role as an ATPase inhib< Control 1.5-> / Coy 1.5 itor, DCCD has been shown to decrease the backflux ofprotons in Halobacterium halobium (21) and in certain ATPaseless mutants of E. coli (1). A similar effect of DCCD on E. halophila could account for the stimulation in proline uptake. The retardation of proton influx CCP =C=;C L O would increase the magnitude of the pH gradient. It is not obvious whether Ap would be 12 8 4 0 increased correspondingly. For example, cation TIME (min) influx could compensate for the decresed influx FIG. 3. Effects of inhibitors on proline uptake. of protons so as to minimize the increase in A+. Cells were illuminated throughout the experiment. However, an indication that Aqf had been inInhibitors were added 5 min before the addition of creased comes from our observation that DCCD [14C]proline at zero time. All inhibitors were dis- causes a slight stimulation in the light-induced solved in ethanol and the control contained a corre- uptake of TPMP+ (see Table 2). sponding level of ethanol (0.2%). Glutamate transport system. The initial rate of glutamate uptake in the light (Fig. 5) is ATP I uptake higher than that observed with proline. Also, bvel (PCfp DCCD .-% the maximum accumulation of glutamate is 3.0 3.0 I attained in a shorter period. The addition of an excess of [12C]glutamate caused a slow efflux of previously accumulated [14C]glutamate. Neither D_[12C]glutamate nor L_[-2C]aspartate promoted efflux of the i_[14C]glutamate (data not c E dk h'IY shown). 1.5 1.5 ak hv Inhibition of glutamate uptake. CCCP or I I i inhibited glutamate uptake by illumiHQNO 11 I 11 0-a nated cells (Fig. 6), but the effects were not as 11 I 9U I pronounced as were seen with proline uptake 0.u c
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1
JouNAiL oF BACTZRIoLOGY, Sept. 1976, p. 1265-1264 Copyright C 1976 American Society for Microbiology
Energy Coupling in the Active Transport of Proline and Glutamate by the Photosynthetic Halophile Ectothiorhodospira halophila CLIFFORD A. RINEHART AND JERRY S. HUBBARD* School ofBiology, Georgia Institute of Technology, Atlanta, Georgia 30332 Received for publication 5 April 1976
When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by darkexposed cells. CCCP strongly inhibited glutamate uptake in the dark. The lightdependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.
Gram-negative organisms have been shown
membrane-bound systems are not yet completely understood. Initially, Kaback and Barnes (14) proposed a working model based on oxidation-reduction coupling at discrete portions of the respiratory chain. Others (2, 7, 11, 16, 21) interpreted their results as supporting Mitchell's chemiosmotic theory of membrane transport (22, 23). This theory states that the energy-transducing reactions of electron transport, ATP synthesis, and active transport are phosphatase (ATPase). The second class is linked energetically and mechanistically by the termed a membrane-bound or respiratory- proton-motive force. The force is derived from linked system. These shock-resistant systems the efflux of protons during electron transport contain permeases but do not contain dissocia- and/or by hydrolysis of ATP by the membrane ble binding proteins. The driving force for the ATPase (2, 4). The proton-motive force (Ap) is second type is not necessarily derived from composed of transmembrane gradients of elecATP. Systems of the latter class have been trical and chemical potentials: Ap = A* extensively studied in membrane vesicles (see ZApH, where A* is the membrane potential (interior negative), ApH is the pH gradient reference 13 for a review). The mechanisms of energy coupling to the (interior alkaline), and Z is a factor to convert
to possess two distinct classes of active transport systems that do not chemically modify the substrate (3, 4, 31). Shock-sensitive systems contain a dissociable binding protein in addition to the permease. These shockable systems are coupled to the phosphate bond energy of adenosine triphosphate (ATP) or a similar high-energy compound by an unknown mechanism not using the membrane adenosine tri-
1255
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RINEHART AND HUBBARD
pH units into electrical units, equaling about 59 mV/pH unit at 250C. These separate components of the proton-motive force have been studied individually by the use of specific ionophores and inhibitors. From these studies Hamilton and Niven (6) formulated a model to explain the driving force for the uptake of various amino acids by Staphylococcus aureus. Basic amino acid uptake was driven by Atp via a uniport mechanism, neutral amino acid uptake was driven by Ap via an H+ symport mechanism, and acidic amino acid uptake was driven by -ZApH via an H+ symport mechanism. Only preliminary characterizations have been made on active transport systems of photosynthetic bacteria. In studies with Chromatium sp. strain D, it was concluded that this organism lacks systems of sufficiently high affinity for efficient amino acid transport (30). The uptake of dicarboxylic acids and pyruvate have been studied in the purple, nonsulfur bacterium Rhodopseudomonas spheroides (5). Hellingwerfet al. (9) recently demonstrated an alanine transport system in cells and membrane vesicles of R. spheroides. The energy to drive alanine uptake could be derived from aerobic respiration in the dark or from anaerobic, photosynthetic electron transport. To our knowledge active transport has not been demonstrated in obligately photosynthetic bacteria. Ectothiorhodospira halophila was originally isolated from a salt pond in southern Oregon by Raymond and Sistrom (27, 28). This purple, sulfur bacterium is obligately anaerobic and photosynthetic. E. halophila is also obligately halophilic, requiring at least 9% NaCl for growth. The species grows well between 11 and 22% NaCl and will grow slowly at 30% NaCl. The photosynthetic apparatus in E. halophila is contained in a complex system of lamellar membranes (27). The objectives of the present study were to characterize the proline and glutamate transport systems in E. halophila and to define the mechanism(s) by which energy is coupled to these processes. MATERIALS AND METHODS Cell suspensions. The culture ofE. halophila was generously provided by W. R. Sistrom. The anaerobic, photosynthetic growth conditions were similar to those previously described (27). A single exception was that our medium contained 0.2% sodium acetate instead of succinate. Thiosulfate was used as the source of reduced sulfur. A 20% NaCl level was used throughout. Cultures were grown in 168-ml milk dilution bottles which were completely filled. A stock culture was stored at 4°C. Cultures were incubated at 41°C under incandescent light for 2 days.
J. BACTERIOL.
Cultures were harvested by centrifugation, washed two times in a buffer of 1.0 M NaCl and 0.02 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8.0, and resuspended in the same buffer to a turbidity of 150 Klett units in a Klett-Summerson colorimeter equipped with a no. 66 filter. This turbidity corresponds to 0.41 mg of cell protein per ml. All harvesting and washing operations were performed at 40C. The cells were kept on ice under ordinary lab light prior to the transport assays. The cells retained approximately 50% of their activity for the uptake of proline and glutamate 24 h after harvesting. Amino acid uptake. Cell suspensions were incubated in glass tubes (16 by 100 mm) at 41.5°C in a Braun Warburg apparatus equipped with incandescent lamps to the exterior of the plexiglass bath. The light intensity reaching the tubes was about 2 mW/ cm2 as measured with a Kettering YSI model 65 radiometer. Dark incubations were conducted in aluminum foil-covered tubes under laboratory lights reduced as far as practical. Unless otherwise indicated, all incubations were conducted with N2 bubbling through the medium at about 80 cm3/min. After a preincubation of 5 min 14C-labeled amino acid was added to the cell suspension and 0.5-ml portions were removed at intervals. The cells were rapidly collected on membrane filters (Gelman GA6, 0.47 jAm, 25-mm diameter) and washed with 1 ml of chilled Tris-1 M NaCl buffer. The filter disks were then placed in scintillation vials and dissolved in a solution containing 1 ml of water, 2 ml of methyl cellosolve, and 10 ml of a cocktail made of dioxane, 10% naphthalene (wt/vol), and 0.5% diphenyloxazole (wt/vol). Samples were counted on a Beckmann LS100C liquid scintillation counter. Unless otherwise indicated, the "4C-labeled amino acids were diluted in 0.1 mM 12C-labeled amino acid and added at levels to give a final concentration of 2 ,uM in the incubation mixture. Initial rates were determined over the 4-min interval immediately after "4C-labeled amino acid addition. All measurements were corrected for retention of "4C-labeled amino acid by the filters in zero time controls. The retention values were 0.09 nmol for [14C]glutamate and 0.05 nmol for
['4C]proline. Uptake of the [3H]triphenylmethylphosphonium cation ([3H]TPMP+) was assayed in a similar manner as described for the amino acids. The membrane filters retained 0.8 nmol of TPMP+ in zero-time controls. Membrane vesicles. Cells suspended in 0.02 M Tris buffer containing 20% sucrose and 0.01 M ethylenediaminetetracetic acid were treated with muramidase (0.5 mg/ml) for 20 min at 23°C. The resulting spheroplasts were then centrifuged and resuspended in the Tris-1 M NaCl buffer to which was added 10 mM sodium ascorbate and 20 ,uM dichloroindophenol to maintain the recommended redox potential (9). Membrane vesicles were then prepared by sonic treatment of the spheroplasts for four 30-s intervals with a Heat Systems Co. Sonifier Cell Disrupter equipped with a microtip. The preparation was incubated with deoxyribonuclease (0.1 mg/ ml) for 15 min to reduce the viscosity and then
VOL. 127, 1976
AMINO ACID TRANSPORT BY E. HALOPHILA
centrifuged for 20 min at 5,000 x g to remove any remaining whole cells. The vesicles were then harvested by centrifugation for 20 min at 45,000 x g and resuspended using a syringe equipped with a 21gauge needle. The preparation was examined microscopically to ascertain that no whole cells remained. In some experiments the intravesicular salt composition was varied by sonically treating the spheroplasts in buffer containing NaCl-KCl mixtures. All preparative operations were at 4 to 5°C. Analytical. Thin-layer chromatography of amino acids was done on silica gel plates (60F-254) obtained from EM Laboratories. The solvent systems were sec-butyl alcohol-formic acid-water (7:1:2) and chloroform-methanol-17% NH4OH solution (4:4:2). Autoradiography was accomplished by exposing du Pont Cronex medical X-ray film to the thin-layer plates. Enzymatic determination of glutamic acid was made by using glutamate decarboxylase prepared from dried cells of Escherichia coli Crookes strain (ATCC 8739). Protein was determined on portions of cells lysed in 0.02 M Tris buffer, using the method of Lowry et al. (20) with bovine serum albumin as the standard. Intracellular ATP was extracted by injecting 0.1 ml of cell suspension into 0.9 ml of boiling 0.02 M Tris, pH 7.4, and the boiling was continued for an additional 2 min. The ATP content of the extract was determined by using the firefly assay as previously reported (11). Chemicals. i_[U-14C]proline (specific activity, 232 Ci/mol) and L_[U-14C]glutamic acid (specific activity, 290 Ci/mol) were obtained from New England Nuclear Corp. [1-14C]glutamic acid (specific activity, 4.84 Ci/mol) was from Calbiochem. Tritiated triphenylmethylphosphonium bromide (specific activity, 114 Ci/mol) was a gift from H. R. Kaback. Unlabeled triphenylmethylphosphonium bromide (K & K Laboratories) was used in a final concentration of 1 mM as an inhibitor and at 0.02 mM as a carrier for the labeled TPMP+. N,N'-dicyclohexylcarbodiimide (DCCD) was from Eastman and was used in a 0.1 mM final concentration. Carbonylcyanide-m-chlorophenylhydrazone (CCCP), used at a 0.01 mM final concentration, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), used at a 0.1 mM final concentration, and valinomycin, used at a 2-,g/ml final concentration, were all from Sigma. The purified and stabilized luciferin-luciferase mixture was from du Pont. All other chemicals were reagent grade and were obtained from standard sources.
RESULTS General aspects of glutamate and proline uptake. Although E. halophila will not grow in media containing less than 1.6 M NaCl, the maximum rate of glutamate uptake was effected by cells suspended in 1 M NaCl (data not shown). The initial rate of ['4C]proline uptake was highest with cells in 1 to 2 M NaCl (data not shown). Thus, 1 M NaCl was chosen as the salt concentration for both glutamate and proline assays so as to achieve highest activities and to simplify preparative procedures.
1257
Several experiments were performed to ascertain that the amino acids were actually transported against a concentration gradient and not chemically altered by the cells. Analyses were made on 14C-labeled amino acids that had been taken up by the cells and then recovered by lysing the cells with distilled water. In the case of [14C]proline the lysate was evaporated and then extracted with absolute ethanol. Over 95% of the total 14C was recovered in this ethanol-soluble fraction. Thin-layer chromatographic and autoradiographic analysis showed that over 90% of the ethanol-soluble 14C was contained in one spot which exhibited chromatographic behavior identical to authentic proline. For the analysis of glutamic acid the cells were allowed to take up [1-14C]glutamate and then lysed as above. Essentially all of the 14C was recovered in the watersoluble fraction. This 14C-labeled extract was mixed with _[12C]glutamic acid and then treated with glutamate decarboxylase, an enzyme which specifically cleaves the number one carbon of i-glutamate. The CO2 evolved was trapped in hyamine hydroxide and its radioactivity was determined. The finding that the "4Clabeled fraction recovered from the cells was decarboxylated to the same extent as authentic [1-_4C]glutamic acid was evidence that the accumulated amino acid had not been chemically altered. From rough calculations it is apparent that the uptake of both glutamate and proline are concentrative. For example, in typical experiments about half (1 nmol) of the available amino acid (2 nmol/ml) is accumulated. Assuming that the intracellular volume is 80% of the total cell volume of 4.1 ,ul/ml, then 1 nmol of amino acid is contained in 3.3 ,ul of the intracellular space. Thus, the concentration of glutamate or proline is about 300 times greater inside the cells than outside. The uptake of both proline and glutamate were inhibited by bubbling 02 through the incubation mixture or by increasing the light intensity to 30 mW/cm2 (data not shown). As will be shown below, E. halophila cells take up glutamate in the dark. Several lines of evidence contraindicate the possibility that this activity reflects some unknown form of dark respiration whereby electrons are transferred to an organic or inorganic acceptor. The uptake of glutamate in the dark was not stimulated by providing oxygen; in fact, it was inhibited. Also, neither thiosulfate nor acetate stimulated the dark activity under anaerobic conditions. This activity was not enhanced by providing the electron donors ascorbate plus phenazine methosulfate or ascorbate plus N,N,N',N'-tet-
1258
J. BACTERIOL.
RINEHART AND HUBBARD
ramethyl-1,4-phenyldiamine (TMPD). The use of phenazine methosulfate, TMPD, or methyl viologen as potential electron acceptors did not stimulate this dark activity. Evidence is presented below that this dark process is driven by the hydrolysis of ATP by membrane ATPase. We also excluded the possibility that dissociable binding proteins were involved in the proline or glutamate transport systems. Subjecting cells or spheroplasts to rapid changes in osmotic strength (1 to 3.4 M NaCl) did not inactivate either system. Also, the observation of active systems in membrane vesicle preparations (see Fig. 8) argues against dissociable components being parts of the transport systems. Proline transport system. Proline uptake by illuminated cells proceeds until the maximum intracellular concentration is approached at about 30 min (Fig. 1). The uptake does not occur in the dark, nor is the uptake stimulated by preincubating the cells in the light, placing them in the dark, and immediately adding [14C]proline. An efflux of [14C]proline occurs when an excess of [12C]proline is added to the incubation after the cells have previously accumulated ['4C]proline. A loss of ['4C]proline from the cells also occurs when the light is turned off (Fig. 1). Photophosphorylation and proline uptake. The intracellular ATP reaches a maximum level of about 3.5 nmol/mg of cell protein in less than 5 min of incubation in the light (Fig. 2). This level is maintained during the course of proline uptake in the light. A slow drop in the intracellular ATP level occurs when illumination is discontinued. Although the cells initially contained as much ATP in the dark (after preillumination) as in irradiated cells, this amount was not associated with proline uptake. Inhibition of proline uptake. CCCP, a compound which renders membranes permeable to protons (24) and uncouples photophosphorylation (12), almost completely inhibits proline uptake (Fig. 3). HQNO, an inhibitor of photosynthetic electron transport (12), is also a strong inhibitor. The ATPase inhibitor (8) DCCD stimulates proline accumulation. In Fig. 4 the time course of proline uptake and ATP levels were compared in the presence of DCCD or CCCP. The addition of DCCD causes a drop in the intracellular ATP level, while the uptake of [14C]proline proceeds rapidly. In contrast, with CCCP present ATP was maintained at the initial level and proline uptake was almost abolished. The maintenance of an intermediate level of ATP in cells exposed to CCCP suggests that some photophosphoryla-
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TIME, min FIG. 1. Proline uptake and efflux in the light and dark. Symbols: 0, standard conditions in light; O, incubated in light for 10 min and then made dark, and the incubation was continued; 0, incubated in light for 10 min, 2 mM ["2C]proline was then added, and the light incubation was continued; A, dark throughout; A, after 5 min of preincubation in the light, the tubes were placed in the dark concomitant with the addition of ['4C]proline at zero time. ATP F 14C-pro I level LSuptakeE
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-
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FIG. 2. Time course of proline uptake and intracellular ATP levels. hp = cells illuminated throughout; hi, -- dK = cells were preilluminated for 5 min and then placed in the dark. ['4C]proline was added at zero time in both experiments.
tion was occurring. If CCCP had completely blocked photophosphorylation, the consuming reactions of the cell should deplete the ATP as was observed with DCCDYpresent (Fig. 4). We cannot exclude the possibility that the ATP formation in the presence of CCCP was occurring by substrate level phosphorylation, but
AMINO ACID TRANSPORT BY E. HALOPHILA
VOL. 127, 1976
1259
line). Thus, the inhibition of proline uptake by TPMP+ suggests that Aq, is an essential component of the driving force. From the findings of Fig. 2 and 4 it is apparent that proline uptake is not directly related to the ATP level of the cells. Moreover, the results with DCCD contraindicated ATP hydrolysis as being of importance in providing the driving force. In addition to its role as an ATPase inhib< Control 1.5-> / Coy 1.5 itor, DCCD has been shown to decrease the backflux ofprotons in Halobacterium halobium (21) and in certain ATPaseless mutants of E. coli (1). A similar effect of DCCD on E. halophila could account for the stimulation in proline uptake. The retardation of proton influx CCP =C=;C L O would increase the magnitude of the pH gradient. It is not obvious whether Ap would be 12 8 4 0 increased correspondingly. For example, cation TIME (min) influx could compensate for the decresed influx FIG. 3. Effects of inhibitors on proline uptake. of protons so as to minimize the increase in A+. Cells were illuminated throughout the experiment. However, an indication that Aqf had been inInhibitors were added 5 min before the addition of creased comes from our observation that DCCD [14C]proline at zero time. All inhibitors were dis- causes a slight stimulation in the light-induced solved in ethanol and the control contained a corre- uptake of TPMP+ (see Table 2). sponding level of ethanol (0.2%). Glutamate transport system. The initial rate of glutamate uptake in the light (Fig. 5) is ATP I uptake higher than that observed with proline. Also, bvel (PCfp DCCD .-% the maximum accumulation of glutamate is 3.0 3.0 I attained in a shorter period. The addition of an excess of [12C]glutamate caused a slow efflux of previously accumulated [14C]glutamate. Neither D_[12C]glutamate nor L_[-2C]aspartate promoted efflux of the i_[14C]glutamate (data not c E dk h'IY shown). 1.5 1.5 ak hv Inhibition of glutamate uptake. CCCP or I I i inhibited glutamate uptake by illumiHQNO 11 I 11 0-a nated cells (Fig. 6), but the effects were not as 11 I 9U I pronounced as were seen with proline uptake 0.u c
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