Efficiency of Light-Driven Metabolite Transport in the Photosynthetic

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Aug 13, 1981 - An evaluation of the efficiency of the L-alanine and L-malate transport systems was undertaken with the photosynthetic bacterium ...
JOURNAL OF BACTERIOLOGY, June 1982, p. 1322-1328 0021-9193/82/061322$07S2.0O)

Vol. 150, No. 3

Efficiency of Light-Driven Metabolite Transport in the Photosynthetic Bacterium Rhodospirillum rubrum MICHAEL ZEBROWER AND PAUL A. LOACH* Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60201 Received 13 August 1981/Accepted 28 January 1982

An evaluation of the efficiency of the L-alanine and L-malate transport systems was undertaken with the photosynthetic bacterium Rhodospirillum rubrum grown on the amino acid whose uptake was measured. An all-glass apparatus was constructed for measuring transport activity under anaerobic conditions. LAlanine transport activity decreased under conditions of Mg2e depletion. When cells were allowed to become inactive by suspending them in the dark in Mg2free buffer, full activity could be restored within a few minutes by adding 20 mM Mg2+ and illuminating the cells. The transport activity was completely inhibited by carbonyl cyanide m-trifluoromethoxyphenylhydrazone and by ammonia. The quantum yield for the uptake of either L-alanine or L-malate was 0.015 molecules per photon. The results are discussed in relation to the expected efficiencies for metabolite transport and regulation by Mg2+. Although light-dependent transport of metabolites has been characterized to some extent in several photosynthetic bacteria (2, 8, 9, 11, 16), the overall efficiency of light-dependent transport of metabolites has not yet been examined in bacteriochlorophyll-containing photosynthetic organisms. In nonphotosynthetic bacteria which grow aerobically, the percentage of the energy released by electron transport which is coupled to transport of amino acids is small. For example, in Bacillus subtilis, one molecule of serine is taken up for approximately 200 to 4,000 electrons passing from substrate to molecular oxygen (13). Evidence exists which indicates that in photosynthetic bacteria (8-10, 22), as well as in many nonphotosynthetic bacteria, metabolite transport systems may function by directly using an electrochemical gradient without first making ATP. On the other hand, in photosynthetic organisms such as plants and algae (15), as well as in many bacteria (23), the direct use of ATP to drive the transport of necessary metabolites has been reported. By studying metabolite transport under light-limiting conditions, as is necessary for quantum yield measurements, we hope to learn more about how these transport systems are coupled to the primary photochemical event and to secondary electron transport. MATERILS AND METHODS Growth and harvest of bacteria. Rhodospirillum rubrum (no. 1.1.1.), originally obtained from R. Y. Stanier, was grown anaerobically on two media. The first was modified Hutner medium (4), which contains 20 mM ammonium malate and 1 g of casein hydroly-

zate per liter as carbon and nitrogen sources. In the second medium these were replaced by 16 mM alanine. The light intensity used during growth was 3 J/m2 per s and was provided by Westinghouse F40W fluorescent lamps. The temperature was maintained between 27 and 29°C. The starter cultures, which were periodically prepared from lyophilized cells stored at -20°C, were kept for 1 day in the dark to deplete the concentration of residual oxygen in the medium before being placed in the light box for 3 additional days. At the end of this period, a 7% inoculum was transferred into fresh medium, and the growth cycle was repeated. Samples used in measuring the rate of light-driven transport were centrifuged at high speed in a table-top centrifuge for 15 min, and the pellets were suspended in about 70 ml of modified Hutner medium containing 20 mM MgSO4 but lacking a carbon source. The cells were then illuminated for 30 min before being centrifuged and suspended in 20 mM potassium phosphate buffer (pH 6.8) containing 20 mM MgSO4. The cell concentration was adjusted to give an absorbance of 1.3 at 880 nm. Radioactive materials. All radioactive compounds used were obtained from New England Nuclear Corp., Boston, Mass. Stock solutions of L-[14C]alanine, D['4C]alanine, and L-[14C]malate were prepared to contain 2.5 pXCi/Lmol. Ight Intensity meaurements. The light source was a 1,000 W tungsten projection lamp controlled by a variable transformer. The light was collimated with two lenses and passed through a 5-cm path of deionized water and either one or two Corning glass 7-69 ifiters. Each of these ifiters has a transparency of 75% at 865 nm. The intensity of the light was measured with an Eppley thermopile which had an eight-junction bismuth-silver circular surface coated with lampblack with a basic sensitivity of 0.118 V/W per cm2 with a quartz window. A Keithley model 149 milli-microvoltmeter was used to measure the voltages produced at

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the thermopile junction. The voltages used for calibra- cell was constructed from a Markson 32G-20 spectrotion were the averages of several measurements as photometer cell. The Teflon plug of the cell had five recorded on a Mosley X-Y recorder (model 2D-2A). A holes drilled into it. Four of these were filled with few experiments were conducted with flash illumina- stainless steel needles (held in place with Eastman 910 tion with a Sunpack Thyristor 611 flash lamp at a adhesive) having the following functions: liquid in, 22G needle; liquid out, 18G needle; gas in, 18G needle; setting of 1/128. To calculate the actual amount of light absorbed by gas out, 13G needle. The needles were located in the a sample, we measured the light intensity through an cell so as to have the minimum interference possible empty illumination cell and then through an illumina- on the light beam passing through the cell. The fifth tion cell containing 3 ml of cell suspension without the hole was covered at both ends with a small rubber needle assembly in place. The difference in the intensi- septum which was also held in place by Eastman 910 ties was then adjusted by the correction factor as adhesive. The radioactive substrate was injected outlined below. A ferrioxalate actinometer solution through this septum. A thin coat of Dow Corning high was used to determine the effects of the various vacuum grease was used to help seal the Teflon plug in stainless steel needles present within the illumination the illumination cell. The relationship of the cell to the cell on the actual amount of light absorbed by the rest of the apparatus was such that there was always sample. Crystalline ferrioxalate was prepared by the positive pressure exerted on the plug to keep it in method of Hatchard and Parker (7). Ferrous ion result- place, even when the internal pressure increased due ing from exposure of the ferrioxalate to 365-nm excit- to the bubbling of argon. The ability ofthe illumination ing light was measured by the o-phenanthroline assay. cell to maintain an anaerobic environment was verified The absorbance of the ferrioxalate solution was adjust- periodically by testing with redox dyes (e.g., indigoteted to 1.3 at 365 nm, since this was the absorbance at rasulfonic acid), which in their reduced forms are quite 880 nm used for the R. rubrum cell suspensions. At sensitive to oxygen. The remainder of the anaerobic transport apparatus this absorbance the value for the quantum yield of the ferrioxalate actinometer is 1.18 (7). Our measured was constructed entirely of glass and was designed to values were 1.13 ± 0.06 (six determinations) for the allow continuous flushing with argon of both the cell illumination cell without the needle assembly in place suspension and the washing buffer. Water traps were and 1.52 ± 0.08 (six determinations) for the cell with placed at the exit ports to minimize back leakage of air the needle assembly in place. Thus, a correction factor into the argon lines. A series of three-way valves was of 1.34 was used to correct for what was presumed to used to connect the cell suspension and washing buffer be primarily a reflection effect of the needles that to two syringes. These syringes were used to measure caused more light to be absorbed in their presence. No the amount of cell suspension or washing buffer to be correction factor was found necessary for the effect of used in each assay. Another set of three-way valves the bubbling of water-saturated argon through the cell connected the syringes to the illumination cell and suspension. filter assembly. The ifiter assembly was designed so Anaerobic appar for n rt assays. A sche- that the filter could be removed without disturbing the matic diagram of the anaerobic apparatus used for anaerobicity of the illumination cell. Once a new ifiter transport assays is shown in Fig. 1. The illumination was in place, the vacuum line, which had been ex-

Measuring Syringe

FIG. 1. Anaerobic transport apparatus. Details are explained in the text.

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posed to air, could be flushed with argon, thus reestablishing the anaerobicity of the entire system. Transport assay. The bacteria were placed in the degassing bottle of the apparatus and degassed for 1 h with prepurified argon obtained from Matheson Scientific, Inc., Elk Grove Village, Ill. By adjusting the appropriate three-way valves and the argon pressure, 3 ml of cell suspension was anaerobically transferred to the illumination cell. The sample was degassed in the illumination cell for an additional 7 min, and then radioactive substrate was injected through the septum of the illumination cell to give a final concentration of 16.5 ,uM. This was well above the saturating substrate level of L-alanine as indicated by experiments in which the substrate concentration was varied. The sample was incubated for a specified amount of time, either under illumination or in the dark. The three-way valves were used to adjust the argon pressure to cause the sample to be filtered through the 1.2-,um pore size Millipore ifiter; this procedure required 6 to 7 s. The ifiter was then washed anaerobically with 20 mM phosphate buffer (pH 6.8), removed, and dried. The illumination cell was washed out with 20 mM anaerobic phosphate buffer in preparation for the next sample.

RESULTS

Effect of air on trnsport activity. High rates of L-malate transport occurred in the dark when air was present (Table 1). A greater than 10-fold reduction in L-malate uptake in the dark was observed when oxygen was removed, but the light-activated uptake remained at a high level. The high level of dark uptake in the presence of air, presumably due to energy made available by aerobic electron transport, was completely inhibited by carbonyl cyanide m-trifluoromethoxyphenylhydrazone or ammonia, as was the lightdependent activity (data not shown). High levels of dark uptake in air were also observed with Lalanine in cells grown on this metabolite. Stabilization of the system aginst loss of trnsport activity. At the low levels of light used in the quantum yield determinations, the cell suspensions showed a pronounced decrease in their rates of L-alanine uptake during storage in the dark in 20 mM potassium phosphate buffer (pH 6.8) which contained no divalent metal cations (Fig. 2A). The addition of Mg2+ had two effects: it stabilized transport activity and increased the maximal rate of transport observed under these conditions (Fig. 2). At high Mg2+ concentrations TABLE 1. Effect of oxygen on L-malate transport Malate transport (nmol/min per mg of celis) Condition

.

Aerobic

Semianaerobic

A

Anaerobic

2.7 12.6 10.8 0.7 6.7 7.2 Dark a All experiments were conducted under saturating light conditions.

Lighta

a fivefold difference in rate was observed between cell suspensions depleted of Mg2e for 2 h and cell suspensions containing Mg2+. Having stabilized the rate of light-dependent uptake, we were able to conduct accurate assays in which the total time of exposure to light could be varied at a specific light intensity (Fig. 3). The time dependence of L-alanine uptake when the system remained dark and anaerobic is also shown (Fig. 3). Note that the dark transport occurred very rapidly and then was essentially constant after 20 s. There was no lag between the onset of L-alanine uptake and the incidence of light, within the limits of our ability to make the first measurements (Fig. 3). Furthermore, the rate of transport was constant for at least the first 2 min after the light was turned on. During an illumination period of about 4 min under saturating light conditions, about 25% of the total L-alanine present was taken up before no further net uptake was observed (Fig. 3). At this point, the concentration ratio of L-alanine (or metabolized products) inside the cell to Lalanine outside the cell was approximately 400:1. Cells which accumulated metabolite in the light lost most of it within about 30 min after the light was turned off (data not shown). This loss of radioactive alanine was greatly accelerated (90%o lost in 5 min) by the addition of a large excess of unlabeled alanine, which presumably can exchange with the L-[14C]alanine inside the cell. Attempts to opdtmi the transport rate. Various Na+, KV, or phosphate concentrations had little effect on the rate of light-dependent transport. Replacing K+ by Na+ had no effect, nor did the transport system seem to be sensitive to the ionic strength of the cell suspension when the concentrations of the buffer components were changed. In addition, there was less than 8% variation in the rate of transport under low intensities of absorbed light (1.4 x 10"5 photons per s) in experiments conducted at pH 5.8, 6.8, and 7.8. The rate of transport observed in cultures grown for 2 or 4 days was identical to that observed in cultures grown for 1 day when the rates were normalized to the bacteriochlorophyll contents of the cultures. Varying the length of the starvation step (suspension and illumination of pelleted cells in buffer with Mg2+ but without L-alanine) also did not have any appreciable effect. Thus, under saturating light conditions, the optimal Km for L-alanine uptake was 1.7 ,uM, and the VXn was 2.1 nmol/min per mg of cells. When D-[14C]alanine was substituted for L[14C]alanine, the same rate of uptake was observed. Observations on the transport of D- and L-alanine in nonphotosynthetic bacteria (e.g., Streptococcus faecalis) indicate that D-alanine

VoL. 150, 1982

METABOLITE TRANSPORT IN R. RUBRUM A

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FIG. 2. (A) Rate of L-alanine uptake in whole celis of R. rubrum as a function of dark incubation time before assay. The light intensity absorbed by the sample was 5.9 x 1015 photons per s. The net light-dependent uptake was measured after a 2-min exposure to light after the addition of 16 ,M L-[(4C]alanine in the absence (A) and presence (0) of 20 mM (B) Dependence of the rate of L-alamnne uptake on Mg2+ concentration. The light intensity absorbed by each sample was 1.4 x 1015 photons per s. Each point represents the rate of uptake

Mg2e.

measured by using data from the first 2 min at the specified Mg2" concentration obtained after 135 min of dark incubation during degassing.

can act as a competitive inhibitor of L-alanine uptake, as well as being taken up itself (20, 24). As might be expected, the maximal rate of Lalanine uptake by cells grown on D,L-malate was

less than 5% of that of L-alanine uptake by cells L-alanine. Effect of uncouplers and electron transport

grown on

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6 8 IlO TIME (min.) FIG. 3. Time dependence of L-alanine uptake upon illumination (0) or in the dark (A) after the addition of L-[(4C]alanine. The light intensity absorbed by each illuminated sample was 1.4 x 1015 photons per s. O

2

4

inhibitors. Dicyclohexylcarbodiimide and oligomycin, both known to be very effective inhibitors of the ATPase in both photosynthetic and nonphotosynthetic organisms (3, 25, 26), were tested for their effects on transport. At concentrations of 167 pg/ml and 160 ,uM, respectively, no effect on the rate of transport was observed at either high (saturating) or low (limiting) light intensity. The uncouplers carbonyl cyanide mtrifluoromethoxyphenylhydrazone and ammonia reduced the rate of light-dependent transport (as well as the dark transport) by more than 90%o at 10 pM and 20 mM, respectively. Antimycin A at 20 ,uM inhibited the uptake rate by 65%. From these results it may be concluded that a functioning cyclic electron transport system capable of sustaining a proton gradient is required for the transport system to work in an efficient manner. Dependence of transport activity on light intensity. The dependence of the rate of L-alanine uptake on light intensity is shown in Fig. 4. The maximal rate of 2.1 nmol/min per mg of cells compares quite favorably with values reported by others who have studied the same metabolite in other photosynthetic (8, 11, 12) and nonphotosynthetic (13) bacteria. Rates for alanine transport in Chromatium vinosum (12) and Rhodopseudomonas sphaeroides (8) are approximately 35% of those seen in this system. Quantum yield measurements. From measurements of the initial rates of alanine uptake and of the light absorbed during continuous illumination, the quantum efficiency for the system could be calculated. After making such measurements at different light intensities and extrapolating to zero light intensity, we obtained a

ZEBROWER AND LOACH

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Reactivation of the inactivated L-alanlne tranport system. When probing the cause of the loss of transport activity in the absence of Mg2", we observed that when the cells were illuminated while they were suspended in phosphate buffer without divalent metal ions, all activity was retained. However, subsequent illumination of cells which had stood in the dark for several hours in the buffer without divalent cations resulted in only a very slow and partial restoration of activity (data not shown). When the cells were treated with EDTA, L-alanine transport activity was rapidly lost whether or not the cells were illuminated during the treatment. The protecting effect of light could be due to the activation of an Mg2+ pump which helped to retain the

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9 12 I (1015 Photons/sec) FIG. 4. Dependence of the initial rate of L-alanine uptake on light intensity. At each value the rate of uptake was measured by using data from the first 2 min. 6

quantum yield of 0.015 t 0.002 molecules per photon for L-alanine uptake (Fig. 5A). We obtained the same value for L-malate uptake (Fig. 5B). A few experiments were also conducted in which a xenon flash lamp was used to excite the sample and the resultant L-alanine transport was measured. The duration of each flash was less than 100 jjs, and the intensity was sufficient to saturate all photosynthetic units. From four measurements with 60, 120, 180, and 240 flashes at the rate of 2 flashes per s, we obtained a quantum yield of 0.014 ± 0.003 molecules per photon.

~~A

.01I 4

Mg2+.

Several experiments were conducted in an attempt to reconstitute L-alanine transport activity in cells that were inactivated. When Mg2+ was added to whole cells which had been exposed to several hours of dark incubation in phosphate buffer without divalent metal cations to inactivate the L-alanine transport system, subsequent measurement of transport showed that no activity was regained even after the cells had stood for 3 or 4 h. However, when the inactive system to which Mg2+ was added was also illuminated with far-red light (800 to 900 nm), complete transport activity was restored within a few minutes under anaerobic conditions (Fig. 6). Approximately 6 quanta absorbed per reaction center was sufficient to restore activity. Regeneration of activity (up to 60%o of maximal) was also found when MnCl2 or CaCl2 was added at 10 mM and the system illuminated at high light intensity. In the latter experiments it was necessary to use 0.02 M HEPES (N-2-hydroxyethylpi-

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Fig. 5. Quantum yield for L-alanine (A) and L-malate (B) uptake measured during the first 2 min of iUumination.

VOL. 150, 1982

METABOLITE TRANSPORT IN R. RUBRUM

E 022's0.96_

,,0.3

0 2 4 6 8 10 12 1416 18 20 Incubation Time in Light (min)

FIG. 6. Rate of L-alanine uptake by whole cells of R. rubrum after inactivation by 2 to 2.5 h of dark incubation in 20 mM potassium phosphate buffer (pH 6.8) containing no Mg" followed by further incubation in light (3.6 x 1014 photons per s per cm2) with 20 mM MgSO4. The dark rate of L-alanine uptake has been subtracted from the data. 1,4-Napthoquinone (20 plM) was added to all samples. The light intensity absorbed by the sample during the assay was 1015 photons per s.

perazine-N'-2-ethanesulfonic acid) buffer (pH 6.8) with 1 mM phosphate to avoid precipitation of the Mn or Ca. Thus, not only, a divalent cation, but also light, which presumably is responsible for the formation of a needed electrochemical gradient, is required for activity. DISCUSSION Our efficiency measurements indicated that one L-alamine, D-alanine, or L-malate molecule was taken up for every 67 quanta absorbed by R. rubrum cells. This efficiency is consistent with the expectation that for each molecule of, for example, L-alanine taken up, a great deal of energy, presumably in the form of ATP, will be required to support the biosynthesis of carbohydrate, protein, lipid, nucleic acid, etc. In the aerobically growing nonphotosynthetic bacterium B. subtilis, one amino acid is taken up for each 200 to 4,000 electrons passing from a substrate molecule to molecular oxygen (13). Thus, the fraction of energy used for metabolite uptake by R. rubrum was significantly greater. It is of interest to note that because we grew our cells under quite low light intensity, the cell membrane differentiated into many tubules which, according to electron micrographs (not shown), rather completely filled the cytoplasmic space. Evidence from other laboratories (14, 19) suggests that the part of the membrane which has proliferated into the cell under these anaerobic and low-light conditions is different in many respects from that portion which is located close to the cell wall. If our quantum yield for transport of 0.015, the previously measured quantum

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yield of 0.95 for light capture by the reaction center (17, 18), and the expectation of translocation of two protons per electron during cyclic electron transport (5, 6, 21) are considered, along with an arbitmry assumption that transport of one L-alanine molecule may be possible by coupling it to the transport of one monovalent cation, then only 1 out of every 133 photosynthetic units might be coupled to metabolite transport. If the photosynthetic units were uniformly distributed throughout the entire membrane, then the fraction that might reside in that part of the membrane next to the cell wall would be only about 1% of the total. Perhaps the metabolite transport systems reside only in this region of the membrane. Thus, direct and highly efficient coupling may occur at locations where the membrane is close to the cell wall. The observation of the reversible loss of transport activity under conditions of Mg2+ depletion and darkness is of particular interest. Because the Mg2+ concentration appears to be involved in regulating the association of light-harvesting chlorophyll complexes in green plant photosynthesis (1, 27) and because many membrane transport systems have been proposed to be active only when associated, one might suggest that Mg2e is required for the appropriate association of the components of the L-alanine transport system in R. rubrum. In other work we showed that the photosynthetic units of R. rubrum reversibly dissociate from a cooperative state and reassociate under exactly the same conditions that were observed in the present study for the reversible inactivation of the Lalanine transport system (28; M. Zebrower, Ph.D. thesis, Northwestern University, Evanston, in., 1982). Such a correspondence could be coincidental in that both systems might have a similar dependence on Mg2+ for activation, or the similarity could indicate that Mg2+ is required on the cytoplasmic side of the membrane to activate both systems and that the properties measured reflect the requirements of an Mg2+ uptake system. Regarding this latter possibility, it should be kept in mind that the apparent Km of the system most likely depends on the light intensity at which the measurements are made, the dark history of the system, and, possibly, the growth conditions. A final thought might be offered regarding the role of such a control system involving Mg2+ and an energized state. Although in the laboratory photosynthetic bacteria are usually grown with continuous illumination, in their natural environment they experience light and dark periods. Thus, in a dark period, in the absence of an energy source, there would be no need to have active transport systems functioning, and indeed there would presumably be a need to prevent

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dissipation ofgradients acquired in the light. The lowering of the internal Mg2+ concentration under dark conditions could provide the switch that controls all light-dependent energy coupling reactions. Thus, the experimental results reported here clearly open the way to designing many additional experiments that should greatly aid our understanding of energy utilization and coupling in the photosynthetic bacteria. ACKNOWLEDGMENTS We thank F. C. Neuhaus ofthe Department of Biochemistry and Molecular Biology, Northwestern University, for the sample of D-[4C]alanine used in our transport studies. This research was supported by grants from the U.S. Public Health Service (GM 11741) and the National Science Foundation (PCM 7816669).

J. BACTERIOL.

13. 14.

15.

16.

17. 18.

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