JOURNAL OF BAcTERioLOGY, Sept. 1976, p. 1188-1196 Copyright ©) 1976 American Society for Microbiology

Vol. 127, No. 3 Printed in U.S.A.

Energy Cost of Galactoside Transport to Escherichia coli DALE R. PURDY' AND ARTHUR L. KOCH* Microbiology Department, Indiana University, Bloomington, Indiana 47401

Received for publication 6 April 1976

Energy reserves of Escherichia coli can be depleted by our previously reported procedure to a level such that even the "downhill" transport of o-nitrophenyl-,8D-galactopyranoside (ONPG) is completely dependent upon the exogenous energy supply. The ONPG concentration is high externally to the cells and is low intracellular because of the action of cytoplasmic B-galactosidase. In the present work, depleted cell suspensions have been infused at low, steady rates with glucose and other energy sources while measurements of transport were being made. Comparing the rate of ONPG transport with the rate of introduction of glucose under conditions where the chosen glucose infusion rate limits transport, we find that 89 molecules of ONPG are transported per molecule of fully oxidized glucose. This transport yield is constant over a 6.5-fold range in rate of glucose addition. This constancy over a range of infusion rates implies that transport is the major cellular function under these special conditions. The yield value of 89 is in agreement with the predictions of 76 from Mitchell's chemiosmotic theory and constitutes an independent proof of its validity, since all the other proposed mechanisms of energy coupling predict much smaller yields. The lag from the start of glucose infusion into the reaction cuvette, to the extrapolated time at which a steady state rate of transport and concomitant hydrolysis are achieved, is short (-1 min). Similarly, the time after the infusion is stopped until the rate of transport returns to the background rate is also short. The latter implies that the energy metabolism is directed almost entirely to transport and/or other ongoing cellular processes and not to repair or renewal of an energy-independent, facilitated diffusion system.

Although it has long been obvious that the accumulation of a substance inside a cell to a higher concentration than that outside the cell requires the expenditure of metabolic energy, it is only recently that we have been able to show that energy expenditure is required even when the transported substance is flowing down its electrochemical gradient (24). A test of this question became possible when sufficiently severe procedures were developed to deplete cells of Escherichia coli of energy reserves. Such energy-depleted cells were not only incapable of carrying out accumulative transport but were also incapable of carrying out transport via the galactoside permease system of o-nitrophenyl,8-D-galactopyranoside (ONPG) down this substance's concentration gradient. Although it was very easy to show an energy requirement for "uphill" accumulation of

thiomethyl-,8-D-galactopyranoside (TMG), ear-

lier experiments to test the energy requirement for "downhill" transport failed because the cells eventually lost their membrane integrity and I Present address: Mathematics Departnent, Indiana University, Bloomington, IN 47401.

transport could no longer be measured. For example, when cells were incubated in the presence of a non-utilizable substrate of the galactoside permease, TMG, with no oxidizable carbon source (22) to dissipate energy reserves, as long as the measurements could be made both uphill TMG accumulation and downhill ONPG transport took place. Evidently, such cells had not become energy limited and therefore could not be used for measurement of the energy requirements for these two types of transport. After the fact we can suggest that pumping TMG is not an effective way to dissipate energy reserves, because subsequent to the initial accumulation of TMG further entry and the concommitant exit during the steadystate "plateau" proceed largely on an energyindependent exchange basis (A. L. Koch and G. Fleming, unpublished observation). The depletion procedure now used depended on the accidental finding that incubation in the presence of a non-utilizable substrate of the phosphotransferase system rapidly depleted the bacteria of the energy required for galactoside transport. This was surprising, since galactoside transport is not believed to be mediated by

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group translocation nor be powered by phosphoenolpyruvate (PEP) as is the phosphotransferase system. After the fact we can suggest that

the success of this new technique rests on the fact that a-methyl-D-glucopyranoside (aMG) is transported and phosphorylated by glucosegrown cells having enzyme complexes II and m for glucose transport (35). Because enzyme complex III also functions as a phosphatase (27), entry and phosphorylation of aMG is followed by dephosphorylation and dissipation of the energy. This creates a high internal concentration of free aMG. Subsequently, any exit of aMG by any mechanism, unless it involves reformation of high-energy bonds, represents an energy loss. This latter does not occur (13, 39). Thus, although both TMG and aMG are accumulated and come to a steady state of equal entry and exit, in the case of TMG, metabolic energy is not dissipated with every entry-exit cycle, and in the case of aMG, it is. Additional features of the new system of energy depletion that may also contribute to its effectiveness is the presence of azide during the incubation. Azide blocks TMG accumulation but stimulates (at least initially) (4, 5, 11) the phosphotransferase system. Azide also blocks the terminal oxidation and would tend to cause the cells to break down their reserve substances fermentatively to meet their energy needs. These fermentation products would be eliminated, for the most part, when the incubation mixture is washed in the next stage of the procedure to remove the aMG and the azide. After a 60-min incubation at 37°C in azide and aMG, we found the cells would regain their ability for uphill transport of TMG and group translocation of aMG as well as the downhill transport ONPG on brief exposure to a utilizable carbon source (24). The present experiments extend these studies by measuring the rate of ONPG transport in cells previously energy depleted, when supplied glucose or )-lactate at continuous, but very slow, limiting rates. The results of this study are consistent with the chemiosmotic theory of Mitchell and not with other mechanisms of energy coupling proposed in the literature. It, therefore, provides a qualitatively different kind of evidence in favor of the chemiosmotic theory than the now overwhelming array of kinds of evidence in the literature on its behalf for certain amino acid and carbohydrate transport systems in prokaryotes. MATERIALS AND METHODS Bacterial strain and media. E. coli ML308 (lacI-Y+Z+A+) was grown at 37°C with forced aeration in M9 medium. Usually the carbon source was

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0.2% glucose sterilized separately, although Dlactate in conjunction with glucose was used in certain experiments (see below). Growth on glucose is necessary to achieve high levels of the glucose phosphotransferase system essential for the energydepletion procedure. Energy depletion. Cells were harvested in the exponential phase of growth before the bacterial density exceeded 0.083 mg (dry weight) per ml. The doubling time was always in the range of 40 to 43 min. The cells were washed twice and resuspended to a concentration of 1 mg (dry weight) per ml. Part of this suspension was assayed for downhill ONPG transport as the unstarved control. To the rest of the suspension 40 mM NaN3 and 20 mM aMG were added, and the suspension was incubated for 60 min at 37°C. Then the suspension was washed once and resuspended in M9 containing 50 ,ug of chloramphenicol per ml at a concentration between 0.10 and 0.45 mg (dry weight) per ml and stored on ice for less than 70 min before measurements were made. All centrifugations were made by quickly accelerating to 16,000 x g in a 50-ml tube in a Sorvall SS-1 centrifuge and then immediately and quickly decelerating. Downhill ONPG transport. For both control and energy-depleted cells the following basic procedure was adopted. It represents the evolution of technique most closely described in Koch (25) and Cecchini and Koch (3). The major difference is that all measurements were made at 37°C. Briefly, 10.0 ml of culture was placed in a cuvette (2 by 2 cm), brought to 37°C in a water bath, placed in a thermostatted cuvette holder in the thermostatted compartment of a Cary model 16 spectrophotometer, and stirred magnetically. The measurement at 420 nm was initiated when 0.8 ml of 25 mM ONPG was added, except as noted. The absorbance was recorded on a chart and also stored in a Wang 720C programable calculator. In this way many precise absorbance measurements could be stored for later analysis by regression methods. The least-square slopes in absorbance units per minute were then divided by the concentration of cells and multiplied by 251.5. This factor incorporates the extinction coefficient of o-nitrophenol at pH 7.0 and dilution factors and the fact that a 2.000-cm light path was used. This computation results in the number of micromoles of ONPG hydrolyzed per gram (dry weight) per minute. This quantity has been designated in vivo cellular hydrolysis in previous communications and must be corrected for permease nonmediated processes leading to the hydrolysis of ONPG. The control for this purpose, routinely run, consisted of adding 11.1 mM neutralized formaldehyde. This completely and quickly abolished permease-mediated transport, leaving unchanged the action of the small amount of /B-galactosidase that had passed through the cytoplasmic membrane as well as the small rate of ONPG penetration of the membrane to the region of excess ,3-galactosidase, by other than the galactoside permease route. Infusion of energy sources. Solutions of glucose or D-lactate were pumped into the stirred cuvette via a fine Teflon tube from a Sage 234-7 syringe pump. The flow rate had been calibrated by a dye dilution

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procedure. The pumping rate was 3.4 ,ul/min; this is so small that correction for dilution was never necessary, since the pumping period was never more than 10 min. For the usual glucose concentration employed, this pumping rate corresponds to 1 to 10 nmol/min, and with the concentration of bacteria usually employed, this corresponded to range of 0.55 to 11.0 umol of glucose per g (dry weight) per min. Although the energy-depleted cells do not become leaky when stored on ice for at. least 6 h, we only stored the suspension for 70 min and completed each run within 20 min.

RESULTS Glucose-grown ML 308, harvested from balanced growth with a doubling time of 40 to 43 min, exhibited a range of rates of in vivo hydrolysis of ONPG at 37°C of 500 to 600 g.mol/g (dry weight) per min. When the cells were energy-depleted as described, the rate of absorbancy increase at 420 nm was the same in the presence or absence of formaldehyde. However, sometimes these rates were as low as the formaldehyde-treated, unstarved control rates of about 60 to 70 umol/g (dry weight) per min, and sometimes they were higher. This discrepancy is also apparent in comparing the work of

Koch (24) with that of Cecchini and Koch (3). We have not yet understood the variation in cultural conditions that leads to this variation in results; however, we believe the correction as applied is always valid. We suggest that the increased formaldehyde control after certain cases of energy-depletion treatment is the result of increased membrane permeability, although the cause of this membrane damage resulting from the starvation procedure is obscure. When single small doses of glucose were added to energy-depleted cells there was a short lag, then the increase in the rate of absorbancy at 420 nm became much larger. Subsequently, it returned to nearly the original rate as the substrate was consumed (24). The increment in absorbance attributable to the ONPG transported in response to the added glucose can be calculated from the magnitude of the step in absorbance at 420 nm versus time curve and converted to a transport yield. It can be seen from Table 1 that the apparent yield is in the neighborhood of 30, which is in good agreement with the previous results (24). If, as might be expected from ecological considerations, transport processes have the highest priority on available energy sources, then, as the ambient glucose concentration decreases, the transport yield should increase to some maximum value. We therefore infused glucose solutions into the reaction cuvette at

constant rates to maintain the glucose concentration low but constant. The rate of infusion was kept suffilciently small so that further increase in transport would result from faster addition. A typical experiment is shown in Fig. 1. The infusion pump was turned on durirg the period enclosed between the two vertical bars. After a lag, L1, the rate of color change came to TABLE 1. Transport yield of single additions of glucose Initial glucose concn (,M)a

Transport yield, step methodb (mol of ONPG/mol of glucose)

5.1 1.25

29.1 35.0 25, 30'

5.0Oc

aConcentration of bacteria ranged from 0.1 to 0.41 mg (dry weight) per ml in the reaction cuvette. b The yield was calculated from the difference in absorbance in the middle of the step between the forward extrapolation of the preglucose administration line and the backward extrapolation of the absorbance curve after it had once again become linear (24) (see Fig. 1). I From reference 24.

I

.5.r1

z

co

-j _

m

TIME (min) FIG. 1. ONPG hydrolysis dependent on energy source infusion in energy-depleted cells. For the batch of cells used in this experiment the prestarvation control had a rate of 670 umol/g (dry weight) per min. This was reduced to 67 by the addition of HCHO. The poststarvation HCHO rate was 104 p,mollg (dry weight) per min. The bacterial concentration was 0.101 mg/ml. The initial rate before the pump was turned on was 104 umol/g (dry weight) per min. During the steady state the rate was 477 ,umol/g (dry weight) per min. The lag, LI, is the time between the start of infusion of energy source and the back-extrapolation of the steady-state rate. The lag, L2, is the time between stopping infusion and the intersection of the extrapolated steady-state rate and the subsequent extrapolated base rate. Also shown in the figure is the graphical way of measuring the yield by the step procedure.

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a steady state. When the infusion pump was turned off, the rate decreased again after a lag, L2, similar to the lag when the infusion pump was turned on. An upper limit to the concentration of glucose present during the steady state can be calculated by assuming that no glucose is consumed or converted into other products until a critical concentration is reached and then the consumption becomes constant and equal to the rate of infusion. Concentrations calculated this way are presented in Table 2. These concentrations are, for the most part, much smaller than those for the previous type of experiment in which a single addition was made. Of course, these estimated steady-state glucose pool levels are gross overestimates, because the glucose is transported, phosphorylated, metabolized, etc., and the free external glucose concentration in the steady state may be orders of magnitude smaller than this calculation indicates. In all the experiments reported in this paper, except as noted, the pumping rate was chosen so that the steady-state rate was at least 50% greater than the basal rate of the energy-depleted cells and no more than 75% ofthe rate of the unstarved cells. Since the unstarved rate

TABLmF 2. Transport yield of continuously infused glucose Steady-state input rate

Apparent steady-state Transport yield, slope glucose)e (JAmol of glu- concn (uAM glu- ONPGmol NGmo lcs) cose/g of dry cos) wt per minp

0.55 1.37 1.37 2.83 2.83 3.55

0.22 0.32 0.49 1.10 1.20 2.20d

86.2 86.8 86.9 95.8 89.8 87.5 88.8 ± 1.5e

The bacterial concentration varied from 0.27 to 0.35 mg (dry weight) per ml in the reaction cuvette, and the rate of glucose addition varied from 1.9 to 9.6 nmol/min. b Apparent steady-state concentration was calculated from the glucose infusion rate and from the lag between the time when the infusion was started and when the steady-state hydrolysis rate was achieved. No doubt this is a gross overestimate (see text). c Experimental slopes, corrected for the slope observed in the absence of energy source, were converted using the bacterial concentration and the molar extinction coefficient of ONPG at pH 7.0 of 2,150. d Determined from the kinetics after the pump was turned off. e Mean ± standard error. a

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was usually 10 times larger than the basal rate, this means that there is usually a five- to sixfold range over which the steady-state rate can be manipulated. Table 2 shows the results with a single batch of cells. It can be seen that over the range of rates of glucose addition per unit amount of cells, the transport yield did not deviate appreciably. It certainly follows that the glucose energy source, under the conditions of these experiments, is used for transport processes in a fixed proportion to its uses for all the processes requiring energy in which these cells are engaged. This conclusion applies over this range of rates of addition and after the initial lag for as long as the infusion pump is left on. In the discussion section we will argue that most ofthe energy is used for galactoside transport. Other energy sources have been used with the energy-depleted cells. In the present report, we will give data for -lactate only. This compound was chosen initially because of the then current interest in its serving as an excellent energy source for transport of various substances in membrane vesicles (14). For experiments with D-lactate, E. coli ML308 was grown on 0.2% DL-lactate. This results in cells with very high levels of lac operon products but low levels of the phosphotransferase system for glucose. Therefore, 1 h before harvest 0.2% glucose was added. The energy-depletion experiments were carried out as above. The 1-h treatment was not adequate to deplete the cells fully, so the background rates were high and higher than the formaldehyde control. Nonetheless, the results of two experiments reported in Table 3 are based on clear differences in background slope, and the slope is limited by the pumping of a solution of -lactate. Finally, Table 4 shows the dependence of the apparent yield on the ONPG concentration. The yield falls with decreasing substrate concentration. One would expect a lower yield at low substrate concentration, because the energy may not be funneled toward galactoside transport as effectively then. With no ONPG, the energy would still be consumed by the cells and be used for other processes. Note that the TABLz 3. Transport yield of continuously infused Dlactate Steady-state input rate Transport yield (mol of (/Lmol of D-lactate/g dry ONPG/mol of )lactate) wt] per min)

7.90a 4.01b

a

23.8

24.4 The bacterial concentration was 0.380 mg/ml.

b The bacterial concentration was 0.374 mg/ml.

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TABLE 4. Transport yield as a function of the ONPG concentrationa Pumping rate of glucose (Amol/g [dry wt] per min)

10.98 5.49 2.74

Transport yield by slope method 1.85 mMc

1.42 mMl

0.962 mMc

44.7 76.1 76.8

41.0 60.3

31.9 42.4

Bacterial concentration 0.091 mg/ml. The rates of glucose infusion were 10, 5, and 2.5 ,umol/min, respectively. c ONPG concentration. b

yields are nearly maximal at the highest concentration employed. This is the standard concentration employed in the other experiments. Also note that the yields are lower in this experiment than in Table 2, presumably because of the faster pumping rates employed.

DISCUSSION Table 2 shows that the yield is independent of infusion rate. The conclusion that can be tentatively drawn is that these energy-depleted cells use virtually all the available energy from the catabolism of the infused substrate for galactoside transport. If there were more urgent "maintenance" demands on energy supply we would expect the yield of ONPG transport to increase as the pumping rate increases, and these other needs could be satisfied. On further increase, one would expect the yield to decrease as an oversupply becomes available, because then the cell would do other (lower priority) things with the extra available metabolic energy. Consistent with this latter expectation, we did find (compare Table 1 with Table 2 or Table 4) that small additions of glucose were less efficiently used for galactoside transport than if glucose is infused continuously at very slow rates. However, the former expectation was not observed; rather, even at the slowest infusion rate we could use the yield did not decrease. This means that transport has toppriority allocation on available energy reserves. As one of us has argued, this is reasonable on ecological grounds (26). There is a lag after the infusion pump is started before a steady-state transport rate is established. For the example shown in Fig. 1 with glucose as substrate, the lag, Ll, was 1.4 min. Presumably the lag time in starting is needed for initial penetration of a molecule of glucose and/or the initial phosphorylation(s) that constitute "pump priming" so that PEP can become plentiful enough so that entry of glucose into energy metabolism is not rate lim-

iting. Other possible.contributing factors to the lag include the time to build up steady-state levels of the intermediates of respiratory pathways. The L, lag is several times longer for the continuous infusion experiment in Fig. 1 than it is for discrete addition of glucose. For example, for the experiment in Table 1 where the initial concentration was 5.1 ,uM the lag was 0.44 min. This suggests that the lag is primarily due to the entry of the very first molecule of glucose into each energy-depleted cell's glycolytic pathway. The fact that the lag, L2, between the time the pump is stopped and the time that the rate of transport drops to the background rate is short (1.3 min in the example ofFig. 1) can only mean that the transport of ONPG requires an ongoing coupling to energy metabolism. For example, if it were assumed that "net" transport requires no metabolic energy per se, (i.e., the transport mechanism is basically a facilitated diffusion mechanism) and that energy is only needed for synthesis or repair of the transport mechanism, then there should be a much longer lag before the cessation of transport after infusion is stopped. On this assumption, the metabolic energy would have been used to renew or repair the transport mechanism so that it could function more or less indefinitely after the energy supply had been removed. It also should be pointed out that the energy consumed does not serve for protein synthesis, because chloramphenicol was always present during the assays. The evidence supplied in this paper that transport ability is quickly lost after infusion is stopped augments the evidence of two of the preceding papers (3, 24) and is convincing support of Kepes' (18) idea that transport via the galactoside permease is obligatorily coupled with energy metabolism. Correspondingly, these papers contradict the previous models of Koch (23) and Fox and Kennedy (6) which have the property of being able to function as a facilitated diffusion system. It is curious that this incorrect feature has been maintained in Kepes' most recent model (20) and by Harold (8) and Hamilton (7). The latter two have reviewed the field as partisans for the chemiosmotic theory. Actually, a symporter or antiporter cannot function as a mechanism for facilitated diffusion unless a path for return of protons is provided as in the work of Cecchini and Koch (3). It can of course serve as a mechanism of exchange across the membrane when there are pools of substrate on both sides, since then there need be no net movement of protons. There is a second potential criticism of the experiments reported here: i.e., that the galac-

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tose produced internally by ONPG cleavage might serve as an energy source. We do not believe this is a factor in these experiments, because the cells had not been induced for galactose metabolism during growth. Nor could they have subsequently become induced for the gal operon by galactose produced by the hydrolysis of ONPG; the assays are short and carried out in the presence of chloramphenicol. It can also be argued that any basal level of galactose enzymes does not contribute in an important way, because the rate of transport returns to nearly the same level after the pump is stopped (Fig. 1) as it was before the pump was started. It would not have stopped had even a small fraction of the galactose produced by /-galactosidase been respired. In fact, if the cells had been grown on galactose initially and were then energy depleted, the cells should spontaneously and irreversibly recover the ability to transport ONPG. During a 15-min incubation with ONPG in the absence of infused energy source, this does not occur. Finally, Jan Rushton (unpublished observation) has recently obtained similar results to ours in a gal- strain. Yet a third potential criticism of our procedure and interpretation is that the galactose produced from the entering ONPG may exchange for a second molecule of ONPG on a nonenergy-requiring exchange basis. To the extent that this occurs our estimates of the transport yield are too high. The purpose of a previous paper (25) was to test this possibility; it was concluded on the basis of stopped flow and other measurements that although counterfilux of this kind does take place, it is quantitively a negligible process under the conditions of the downhill assay. Thus, we feel that we can justify our measurement of transport yield as valid against three kinds of objections: (i) the suggestion that our measurements give the average of a lower unit transport cost together with a repair or synthesis surcharge; (ii) the suggestion that galactose metabolism of the product of ONPG hydrolysis contributes to the energy available for transport; and (iii) that countertransport of the galactose supports continuing ONPG transport. The second and third points do, however, provide probable explanations as to why the observed average transport yield of 89 + 1.5 is somewhat larger than the predicted value of 76. Whereas 89 is the best estimate we have of the yield obtained from a series of runs over a period of several months in which cell conditions seem optimum, the estimate of ±1.5 is an exaggeration of the precision of the method in our hands so far. We have occasionally had runs such as that shown in Fig. 1 where the

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yield appears to be higher (130) and other runs where the yield appears lower, such as the values of 76.1 and 76.8 shown in Table 4 for the slower two ofthe pumping rates. But such high and low results are only sporadic and have not exceeded those limits. We now turn to a consideration of the transport yield for glucose expected for different coupling schemes proposed in the literature, whether or not they have been excluded on other grounds. If complete glucose oxidation resulted in the 38 high-energy phosphate bonds normally associated with metabolism via glycolysis and the tricarboxylic acid cycle and each one caused a single transport event, as is probably the case for glutamine transport (2), then the transport yield would be 38. This is the value expected whether glucose is converted to glucose 6-phosphate by the phosphotransferase system or by a hexokinase action after passive diffusion into the cell. Note that 36 high-energy phosphate bonds instead of the 38 will be obtained in eukaryotic cells with mitochondrial organelles, because the two molecules of cytoplasmic-reduced pyridine nucleotides are less efficiently used for energy production than are the reduced pyridine nucleotide molecules formed within the mitochondrion. Only two phosphorylation sites are passed when reduced nicotinamide adenine dinucleotide is oxidized via the glycerol phosphate shuttle instead of the normal three. The value of 38 is expected to apply to prokaryotes in which all the reduced pyridine is formed on the same side (inside) of the respiratory chaincontaining membrane. Of course, a P/O ratio of 3 is the maximal theoretical value and has only been rarely achieved experimentally (31). There could be other losses; for example, to the degree that the phosphatase action of phosphotransferase enzyme HI for glucose liberates free glucose inside the cytoplasmic membrane (27), an extra high-energy bond would be consumed to regenerate glucose-6-phosphate. Another energy coupling system functioning in prokaryotes is the phosphotransferase system (35). If the phosphotransferase type of mechanism functioned, then the transport yield should be only 1, because glucose metabolism yields but 1 net molecule of PEP via the phosphotransferase entry and metabolism via the glycolytic system. However, the transport yield could be raised to 18.5 if the high-energy phosphates made elsewhere in the Embden-Meyerhof-Parnas pathway and Kreb cycle were converted to PEP by processes whereby phosphate bonds at the level of adenosine 5'-triphosphate were converted to PEP via phosphopyruvate

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carboxykinase. In this process two pyrophosphate bonds are converted into one at the level of PEP. A third mechanism to be considered is that of Kaback and Barnes (15). They suggested that there is a single site in the respiratory chain where the passage of a pair of electrons causes the alternate oxidation and reduction of two sulfhydryl residues. This could then drive concomitant cyclical conformational changes that cause a transport event across the cytoplasmic membrane. Only 10 of the 12 pairs of electrons from glucose oxidation should pass this point. Consequently, the transport yield should be 10. Kaback has not stressed this scheme in his latest publications (14) and in fact has partially adopted the chemiosmotic hypothesis (36). We include it here to be inclusive and because the newer model of Schuldiner and Kaback (36) is still subject to this criticism, because they imagine that only electrons passing a particular portion of the electron chain are capable of powering transport. The remaining class of mechanisms can be classified as involving energy-rich membrane states. There is ample reason to conclude that proline and lactose transport involve energy that need not pass through a form in which it is stored as energy of phosphorylation as shown by Berger (2) and Klein and Boyer (21). We shall only consider here stoichiometries based on the chemiosmotic mechanism. The chemiosmotic mechanism as proposed by West and Mitchell yields two protons moved across the membrane for every site that can result in phosphorylation (38). Basically, therefore, Mitchell's mechanism predicts a yield of 76. This would be reduced to 70 if the energy of PEP, adenosine 5'-triphosphate, and guanine 5'-triphosphate (or inosine 5'-triphosphate) produced by substrate level phosphorylation within the fermentative pathways and Kreb cycle are not converted to charge separation energy at a rate of one high-energy bond per pair of protons. From experimental studies, etc. at the present time the H+/O ratio like the P/O ratio appears to be smaller in some bacterial systems than in mitochondria. This has been shown by Lawford and Haddock (29), Hertzberg and Hinkle (9), and Jones et al (12). It is felt by workers in both of the laboratories of Haddock and Jones that there are only two proton loops in the respiratory chain of E. coli. Perhaps our growth conditions on glucose as the sole carbon source together with forced aeration leads to the development of the additional site. Consequently, our experimental value of about 89 ONPG molecules transported per mol-

J. BACTERIOL.

ecule of glucose consumed is strong evidence in favor of the chemiosmotic mechanism with the number of proton loops equal or greater than that of mitochondria over the other suggested modes of energy coupling. To adhere to one of the other models and accept our results at face value one must additionally assume that the carrier is multivalent, i.e., that more than one molecule of substrate is moved from outside to inside per molecule of the actual high-energy state consumed or that the membrane transport event is not stoichiometrically related at all to consumption of the high-energy intermediary. A similar comparison for the metabolism of D-lactate is as follows. Since normal lactic acid fermentation yields two high-energy phosphate bonds per molecule of glucose but complete oxidation ofthat glucose would yield 38, one would anticipate (38-2)/2 = 18 high-energy phosphate bonds. Similarly one expects 36 translocated protons, 34 translocated protons if the energy of succinyl coenzyme A is not translated into proton movement. Five pairs ofprotons would pass the site proposed by Kaback, and no PEP molecules would be generated. The observed value of 24.5 is less than the 36 or 34 predicted by the chemoiosmotic model, but we have not included the cost of transporting the lactate itself. Up until recently, we have tried to remain open-minded about the nature of the energy coupling to active transport for the case of galactosides in E. coli; we feel that multiple energy mechanisms will obtain in different biological situations, perhaps simultaneously. Recently we have presented strong evidence for the chemiosmotic hypothesis for the galactoside permease of E. coli (3). We find that carbonylcyanide m-chlorophenyl-hydrazone and other proton conductors, at concentrations that block completely accumulation of a non-hydrolyzable thiogalactoside, [14C]TMG, actually stimulates downhill in vivo hydrolysis of ONPG by energydepleted cells. The only interpretation compatible with this is that energy depletion eliminates any charge separation across the membrane and that ONPG transport requires the concomitant transport of a proton which builds a reverse protonmotive force in energy-depleted cells that blocks further transport. Carbonylcyanide m-chlorophenyl-hydrazone serves to short out this reverse potential and therefore allows continuing transport down the electrochemical gradient of the ONPG. This combination of a symporter and a proton conductor, which together permit equilibration across the membrane, we have designated as being a pseudo-facilitated diffusion system. The experiments reported here plus the ex-

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periments of Cecchini and Koch (3), we feel, together with the experiments of West and Mitchell (38) and Hirata et al. (10) and Schuldiner and Kaback's (36) new result, are totally convincing support for the chemiosmotic hypothesis in E. coli. Together with the elegant experiments of Kashket and Wilson (16) with Streptococcus lactis, it must be assumed that proton-linked transport is an important mode of coupling of transport to metabolism in prokaryotes. Finally, we should like to make some general comments on the energy cost of transport. Clearly the more free energy dissipated in the transport process, the larger can be the concentration of substrate on the inside of the cell relative to the outside of the cell. Thus, a maximum ratio could be obtained by the phosphotransferase system, a smaller ratio by the dissipation of an adenosine 5'-triphosphate-level phosphate bond, and yet a lower ratio by a symporter carrying one substrate molecule per proton returned to its original side of the membrane. The theoretical maximum ratios will be diminished in most laboratory and natural circumstances because of outward flow not coupled to the reformation of the energy source and by metabolism internally since substrates are usually nongratuitous. The evolutionary choice of which energy-coupling system is used by a particular organism for a particular substrate will be influenced by the ecology of the organism. The energy dissipation of systems considered range possibly four- to fivefold in actual amount of free energy expended. Models in which only a single site on the respiratory chain (such as Kaback's models) could be variable in energy dissipation depending on other conditions in cells having to do with reduction states of cofactors and phosphorylation levels of adenine nucleotides. Since a free energy change of 1.4 kcal/mol corresponds to a 10-fold change in equilibrium concentration, the phosphotransferase system will be able to extract its substrate from a medium several orders of magnitude poorer in its substrate than can be done by an adenosine 5'-triphosphate-linked system. A symporter mechanism followed by a kinase would have an intermediate ability. In an aerobic environment, E. coli spends very little free energy to pump lactose compared with Staphylococcus aureus, which uses the phosphotransferase system (17, 28, 34). From what was said above the former could transport 76 times more lactose per glucose molecule supplied externally. Anaerobically, the difference is only fourfold. However, the fair comparison is not the basic transport costs, but rather is the expenditures to create two

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molecules of hexose phosphate. S. aureus spends one PEP and one -P; E. coli spends one proton and two -P. The difference in energy cost is not much. The standard free energy on the biochemistry scale for PEP is AG = -14,800 cal/mol and for a proton and a -P is approximately 1.5 x -7,000 cal/mol. Finally, it should be pointed out that it is becoming apparent that some substrates may require both phosphate energy and membrane energy but still result in membrane transport of a species retaining its chemical identity (28). Such substrates, one might speculate, have frequently, in the evolutionary past, been the only available energy sources for the organism and even then available at very low chronic-depleted levels, that it was sufficiently worthwhile to the organism to have created such very powerful concentrative mechanisms. ACKNOWLEDGMENTS Work on this project was supported by the National Science Foundation under grant GB-32115 and by Public Health Service grant AI-09337 from the National Institute of Allergy and Infectious Diseases. We wish to thank C. Houston Wang for expert technical assistance and for help in writing the manuscript. LITERATURE CITED 1. Altendorf, K., H. Hirata, and F. M. Harold. 1975. Accumulation of lipid-soluble ions and of rubidium as indicators of the electrical potential in membrane vesicles of Escherichia coli. J. Biol. Chem. 250:14051412. 2. Berger, E. A. 1973. Different mechanisns of energy coupling for the active transport of proline and glutamine in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 70:1514-1518. 3. Cecchini, G., and A. L. Koch. 1975. Effect of uncouplers on "downhill" 3-galactoside transport in energy-depleted cells of Escherichia coli. J. Bacteriol. 123:187195. 4. Englesberg, E., P. Hoffee, and F. Lamy. 1964. The glucose permease system in bacteria. Biochim. Biophys. Acta 79:337-350. 5. Englesberg, E., J. A. Watson, and P. A. Hoffee. 1961. The glucose effect and the relationship between glucose perinease, acid phosphatase, and glucose resistance. Cold Spring Harbor Symp. Quant. Biol. 26:261275. 6. Fox, C. F., and E. P. Kennedy. 1965. Specific labeling and partial purification of the M protein, a component of the ,B-galactosidase transport system ofEscherichia coli. Proc. Natl. Acad. Sci. U.S.A. 54:891-899. 7. Hamilton, W. A. 1975. Energy coupling in microbial transport. Adv. Microb. Physiol. 12:1-53. 8. Harold, F. M. 1972. Conservation and transformation of energy by bacterial membranes. Bacteriol. Rev. 36:172-238. 9. Hertzberg, E. L., and P. C. Hinkle. 1974. Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli. Biochem. Biophys. Res. Commun. 58:178-184. 10. Hirata, H., K. Altendorf, and F. M. Harold. 1973. Role of an electrical potential in the coupling of metabolic energy to active transport by membrane vesicles of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 70:1804-1808.

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