JouRNAL OF BACEROLOGY, JUlY 1975, p. 187-195Copyright i 1975 American Society for Microbiology

Vol. 123, No. 1Printed in U.S.A.

Effect of Uncouplers on "Downhill" f,-Galactoside Transport inEnergy-Depleted Cells of Escherichia coli

GARY CECCHINII* AND ARTHUR L. KOCH2

Department of Microbiology, Indiana University, Bloomington, Indiana 47401

Received for publication 6 January 1975

Galactoside permease-containing cells of Escherichia coli can be depleted ofenergy reserves so that the "downhill" cellular hydrolysis of o-nitrophenyl-fl-D-galactopyranoside (ONPG) no longer takes place. Treatment of such energy-de-pleted cells with proton-conducting agents such as carbonylcyanide m-chloro-phenylhydrazone results in stimulation of ONPG transport. The same agentslower transport of non-energy-depleted cells towards the same levels that resultfrom stimulation of the energy-depleted cells. Of course, these agents prevent"uphill" accumulation against a concentration gradient under all conditions.Since uncouplers allow normal and energy-depleted cells to assume the samefacilitated transport capability, these results lend support to the chemiosmotichypothesis of Mitchell that comigration of charge is necessary for the transport ofneutral galactosides. Our results imply that a potential favorable to transport ismaintained by metabolism in non-energy-depleted cells, whereas an unfavorablepotential is developed in the initial instant of time when energy-depleted cells aregiven ONPG.

For a number of years it has been known thatenergy poisons such as azide and cyanide blockthe accumulation of thiogalactosides in Esche-richia coli (12). However, these energy poisonsshowed less inhibition of the permease-mediated hydrolysis of the chromogenic sub-strate o-nitrophenyl-j3-D-galactopyranoside(ONPG) than they did for the active uptake ofthe non-utilizable thiogalactosides. Therefore,one of us (12) originally proposed that energymetabolism was facultative and not obligatoryfor ONPG transport. Recently, this concept wasretracted because it was shown (14) that, byproper manipulation of the cells, E. coli couldbe starved so that ONPG transport did notoccur even down a thermodynamic gradient.The present work shows that what these energy-depleted cells cannot do is move ions of one signalone across the membrane.Recent models of energy coupling to transport

have centered on electron flow models of trans-port (9, 10) or the chemiosmotic hypothesisproposed by Mitchell (15, 18). According to thechemiosmotic hypothesis, cells extrude protonsas a result of movement of electrons down the

IPresent address: Department of Biological Chemistry,Medical Science I, The University of Michigan, Ann Arbor,Mich. 48104.

'Temporary address (until August 1975): Division ofBiological and Medical Research, Argonne National Labora-tory, Argonne, Ill. 60439.

respiratory chain or due to the action of amembrane-bound Mg2+Ca2+ adenosine 5'-tri-phosphatase (1, 5, 6, 17). This charge separationresults in a membrane potential and/or a pHgradient; the energy conserved in this totalelectrochemical gradient is then available foruse in active transport processes. There is acertain stoichiometric relationship between thecharge moved across the membrane by itsenergy-requiring process and the number ofunidirectional transport events. The membranemust be essentially impermeable to all ions,including protons, if the energy is to be effec-tively stored. One of the features of this model isthe explanation for the action of uncouplerssuch as carbonylcyanide m-chlorophenylhydra-zone (CCCP) and carbonylcyanide p-tri-fluoromethoxy-phenylhydrazone (FCCP),which are classified as proton conductors. It hasbeen shown that CCCP blocks fully thiogalacto-side accumulation (16); we have confirmed thisand shown that efflux of previously accumu-lated thiogalactoside starts within seconds afterthe agent is introduced (unpublished data). Incontrast, we will show that these compoundsonly partially inhibit ONPG hydrolysis.The proton conductor types of uncouplers are

thought to act as lipid-soluble proton donor-acceptor systems which, bearing charge, cantraverse the lipid phase of the membrane and

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188 CECCHINI AND KOCH

can thus conduct charge across it. Thus, theseuncouplers (4) are thought to inhibit activetransport by collapsing the charge separationand discharging the membrane potential.Hopfer et al. (7) have shown there is a correla-tion between the ability of these compounds touncouple and their effectiveness in increasingthe conductivity of protons in artificial mem-branes.

Therefore, we felt the action of proton con-ductors on energy-depleted cells would be anexcellent physiological system to investigateenergy coupling to transport. The chemiosmotictheory proposes that the thermodynamicallydownhill transport to ONPG in energy-depletedcells containing fl-galactosidase would bestalled by a reverse protonmotive force devel-oped by initial symport and irreversible hydrol-ysis of ONPG. This reverse protonmotive forceshould be shorted out by uncouplers that areproton conductors. It was predicted and foundthat the transport of ONPG could be reac-tivated by proton conductors. The transportbeing driven in this case is not powered by meta-bolic energy but by the free energy of hydrolysisof entering ONPG by the excess of f,-galactosid-ase internal to the cytoplasmic membrane.This reactivation is only partial but gives thesame activity that non-energy-depleted cells arereduced to when their favorable protonmotiveforce is shortened out with the same agents.This we believe is critical evidence in support ofMitchell's hypothesis.

MATERIALS AND METHODSBacterial strains and chemicals. E. coli strains

ML308 (lac I- Y+ Z+ A+), ML30 (I+ Y+ Z+ A+), andML35 (I Y- Z+ A+) were used for most studies.Strains 7 and NR70 were kindly provided by B. P.Rosen. ONPG and CCCP were obtained from SigmaChemical Co. FCCP, tetrachlorotrifluorobenzimida-zole, and tetrachlorosalicylanilide were the respectivegifts of R. K. Togasaki, C. E. Furlong, and F. M.Harold. All uncouplers were dissolved in 95% ethanolbefore use.Media and growth conditions. Minimal M-9 salts

medium (pH 7.0) (2) was used. Carbon sources wereautoclaved separately and added to give a finalconcentration of 0.2%. All cultures were grown at 37 Cwith forced aeration and were harvested in the expo-nential phase of growth. The doubling time on glucosefor all ML strains ranged from 41 to 45 min for allexperiments.Permease assay. The rate of in vivo hydrolysis of

ONPG by intracellular ,B-galactosidase is limited bythe rate of transport of ONPG by the ,B-galactosidepermease under the conditions employed. The rate ofappearance of o-nitrophenol was measured at pH 7.0at 420 nm in a Cary model 16 spectrophotometerequipped with a constant-temperature chamber and

cell housing. All measurements were at 28 C. Theinstrument was equipped with an interface and aHouston "Omnigraphic" XT recorder. The cell holderfor cuvettes (2 by 2 cm) was also adapted so that amagnetic mixing cuvette holder could be utilized asdescribed by Koch (13). The continuous mixing, inaddition to providing aeration of the bacterial suspen-sion, permits accurate measurement to be resumedrapidly instead of waiting for the fluctuations on therecorder due to the light-scattering component of thesignal to subside. This transient response can belargely overcome because the cells are partially ori-ented at all times by the stirring device. All permeaseassays were carried out in the presence of 50 sg ofchloramphenicol per ml. For the permease assay, 5 mlof the bacterial suspension at 28 C was added to acuvette containing 5 ml of M-9 medium also at 28 C.The reference beam of the spectrophotometer con-tained a 1-cm cuvette with 2.5 ml of the undilutedbacterial suspension plus 0.2 ml of M-9, which effec-tively compensates for light scattering due to theconcentrated bacterial suspensions. To start the reac-tion 0.8 ml ofONPG was added to the sample cuvette.Permease assays were usually done at 1.85 mMONPG. Any uncoupler was added simultaneouslywith the ONPG. After the reaction had proceeded to0.1 to 0.3 absorbancy units (420 nm) in the 2-cmcuvette, 0.1 ml of formaldehyde (36.6% solutiondiluted 1:10 in water and neutralized to pH 7.0) wasadded to inhibit the permease. This concentrationessentially inhibits the permease system completely.The rate of formation of ONP was again measured,and the rate of hydrolysis by the formaldehyde controlwas subtracted from the initial rate measurement.This gives the net in vivo rate of hydrolysis correctedfor free enzyme and penetration of ONPG into intactcells by "cryptic pathways." The molar extinctioncoefficient of ONP in the M-9 medium was taken as2,150 when measured in a 1-cm cuvette.

Energy depletion. Cells were harvested in theexponential phase of growth at an absorbancy (420nm) of about 0.5 (0.077 mg/ml). Cells were harvestedby centrifugation and washed once with M-9 medium.Succinate-grown cells had 0.2% glucose added to theculture for one generation prior to harvesting. Thisallows sufficient glucose transport activity to beproduced to transport a-methyl-D-glucopyranoside.All centrifugation and washings were carried out atroom temperature. Each centrifugation and wash wascompleted within 2 to 3 min. The washed cells werethen resuspended in M-9 to an absorbancy (420 nm)of 0.6 (0.094 mg/ml) and aerated with 20 mM a-meth-yl-D-glucopyranoside and 40 mM sodium azide at37 C as described previously (14). Glucose-grown cellswere treated for 60 min and succinate-grown cellswere treated for 90 min. The cells were then harvestedby centrifugation and washed once with M-9 medium,followed by resuspension in M-9 medium plus 50,ug ofchloramphenicol per ml and storage at 0 C untilassayed for permease activity. Energy-depleted cellsare stable for up to 6 h under these conditions, i.e.,they did not become leaky. The cells were not storedfor longer than 4 h in experiments reported in thiscommunication.

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RESULTSEnergy depletion of cells. Figure 1 shows the

effect energy depletion has on the cells ability tohydrolyze ONPG by permease-mediatedsteady-state processes. Curve A represents therate of hydrolysis of ONPG in non-energy-de-pleted cells; curve F represents a series of runsin the presence of formaldehyde. Formaldehydeinactivates the permease, and the residual ac-tivity found in formaldehyde-treated cells rep-resents contributions from so-called cryptictransport plus that from a small amount of,-galactosidase outside of the cytoplasmicmembrane. Formaldehyde was added directlyto the reaction cuvette to stop permease-mediated hydrolysis. The recording was contin-ued, and the residual "cryptic" hydrolysis ratewas subtracted from the initial rate to obtainthe "net" hydrolysis rate. The same results wereobtained for formaldehyde-treated cellswhether the formaldehyde was added togetherwith the ONPG or after an interval of time inthe presence of ONPG. In Fig. 1 (curves E andF), formaldehyde was added together withONPG at the start of the assay. All otherformaldehyde-treated cells reported in thispaper had the formaldehyde added to the reac-tion cuvette subsequent to measurement of

1250 A/Bt

1000

E~~~~~~~~~TM (min

0

E 50-

.j20

TIME (min)

FIG. 1. ONPG transport in glucose-grown strainML308. The curves are traced directly from theoriginal recorder tracings and have only beensmoothed to remove instrumental noise, which has aperiod of a few seconds and a magnitude of about 5AmolIg. The curves have been translated so that theygo through the origin. An ordinate scale was chosen tonormalize for the concentration of bacterial cells. (A)Non-energy-depleted cells, which were exponentiallygrowing cells, were washed twice to remove exogenousglucose. (B) Energy-depleted cells treated with 1.1mM glucose for 13 min, washed, and assayed fortransport activity. (C) Energy-depleted cells with 0.1,m glucose added to the stirred cuvette at the timeindicated by the arrow. Concentration of the cells was1.24 mg/mi. (D) Energy-depleted cells. (E) Formalde-hyde-treated energy-depleted cells. (F) Formaldehydecontrol for curves A and B.

ONPG hydrolysis, and these individual rateswere corrected by the rate measured in thepresence of formaldehyde and then designated"net in vivo" hydrolysis. In Fig. 1, curves E andF are the respective controls for the rate ofhydrolysis of energy-depleted and non-energy-depleted cells.

Curve D represents the rate of hydrolysis inenergy-depleted cells. However, by comparingthis to its formaldehyde control (curve E), itmay be seen that the net rate of transport isessentially zero. It must be noted that the ratesof formaldehyde controls for energy-depletedcells were higher than those of non-energy-de-pleted cells (compare curves E and F). This is incontrast to what we have reported previously(14). The reason for the difference has not beenfound. However, controls with levels of thio-digalactopyranoside high enough to inhibit per-mease activity but not sufficient to inhibiteffectively ,B-galactosidase are in good agree-ment with formaldehyde controls (data notshown). Curve B shows that incubating energy-depleted cells with glucose completely reacti-vates the ONPG transport system and lowersthe formaldehyde control rate to that of formal-dehyde-treated non-energy-depleted cells(curve F). Curve C shows that upon addition ofa small amount of glucose to energy-depletedcells the transport system is activated and,when the glucose is used up, the rates return tothose characteristic of energy-depleted cells.The net rate of ONPG transport in glucose-grown ML308 cells is 150 to 200 ,mol/g per min,whereas when they have been energy depletedthis rate falls essentially to zero. The energydepletion has no effect on internal levels off,-galactosidase or the activity of the enzyme(14), since we confirmed that lysed energy-de-pleted cells have the same level of enzyme ascontrol cells.

Effect of proton conductors. It may be seenupon examination of the results with glucose-grown cells (upper curves in Fig. 2 and Table 1)that, when the agents CCCP and FCCP areadded simultaneously with ONPG, transport innon-energy-depleted cells is partially inhibited.It is to be stressed that significant residualactivity remains even though these concentra-tions of CCCP and FCCP used would fully blockthe accumulation of thiogalactosides. The per-cent inhibition of net in vivo hydrolysis is aboutthe same for cells that have been grown onsuccinate (with the addition of glucose for thelast generation) (lower half of Table 1) eventhough all the rates are larger since 3-galactosi-dase and galactoside permease levels are higherin such cells than in catabolite-repressed cells

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grown on glucose alone. The important informa-tion to note in Fig. 2 and Table 1 is that theseuncouplers actually stimulate ONPG transportin energy-depleted cells. This immediately re-moves from the data presented so far thepossible conclusion that these agents have somedirect or indirect action interfering with trans-port. In the case shown in Fig. 2, it is apparentthat the maximum stimulation observed inenergy-depleted cells is not as great as theresidual activity in the presence of uncouplersin non-energy-depleted cells. Even at 20,MCCCP or FCCP there is still a difference ofabout 20,mol/g per min between energy- andnon-energy-depleted cells. This will be dis-cussed further below.The effect on transport of varying the concen-

tration of ONPG is presented in Fig. 3. Atconcentrations of ONPG of less than 2 mm andat a constant high concentration of 20 uMCCCP, the inhibition of transport in non-ener-gy-depleted cells leads to almost identical ratesof net transport (center two lines).Permease-negative strain. With permease-

negative strain ML35 constitutive for ,8-galac-tosidase, proton-conducting uncouplers have noeffect on residual in vivo hydrolysis in eithernon-energy-depleted or energy-depleted cells(Table 2). Although it can be seen that thestarvation procedure rendered the cells slightlyleaky, the experiment shows that it does require

150

0

E 100

0

I 5

w

z

iO

olA I

I

0 5 10 15 20[CCCP] or [FCCP] (PM)

FIG. 2. ONPG transport in glucose-grown strainML308 treated with proton conductors. Data is ex-pressed as net in vivo hydrolysis in which, subsequentto the total rate measurement, a formaldehyde controlwas run on the same sample and subtracted for eachexperimental run. Symbols: 0, non-energy-depletedcells treated with CCCP; 0, such cells treated withFCCP; 0, energy-depleted cells treated with CCCP;U, energy-depleted cells treated with FCCP. The datapoints represent the initial transport rate (30 s to 5min) with uncoupler and ONPG added simultane-ously to initiate the reaction.

TABLE 1. Effect of CCCP on ONPG transport ofglucose and succinate-grown E. coli ML308

Net in vivoONPG

Cells" Assay condition hydrolysis(pmol/gper min)

A. Glucose-grown No addition 183.8non-energy 5MM CCCP 91.4depleted 10MOM CCCP 69.2

Energy depleted No addition 0.05MM CCCP 36.410M4M CCCP 47.42.2MM glucose 102.7

B. Succinate-grown No addition 299.1non-energy 5MM CCCP 140.0depleted 10 MM CCCP 121.0

Energy depleted No addition -0.45 AM CCCP 51.710 AM CCCP 81.07.8,MM succinate 98.8

a Conditions: (A) 0.2% glucose grown; energy de-pleted for 60 min; (B) 0.2% succinate grown with0.2% glucose for last generation; energy depleted for90 min.

a functional permease to observe the stimula-tion of ONPG transport by proton conductors inenergy-depleted cells and that the agents do noteffect the integrity of the cell membrane. Theincreased level of leakiness caused by the energydepletion is small in terms of the measurementof permease-containing cells and, of course, iscompensated for by the formaldehyde control.

Effect in partially induced cells. It was feltthat, if the proton conductors were stimulatingtransport by short circuiting the reverse poten-tial developed by the attempted entry ofONPG, the effect might be enhanced in cellswith lower levels of permease. Cells with lowlevels of permease still have the same area ofmembrane to interact with the uncoupler medi-ating the passage of protons and, therefore, theresistance to the return of the protons would bedecreased relative to the rate that the fewersymporters could move protons from outside toinside. ML30, which is inducible for the lacoperon, was used for the experiments. Culturesgrowing on glucose were minimally induced by apulse of 15 min with 0.5 mM isopropyl-thio-fl-D-galactopyranoside immediately before harvest-ing. In agreement with this supposition, at 20,uM CCCP, stimulation of transport is maximaland even at 10 MM CCCP the difference intransport between non-energy-depleted cellsand energy-depleted cells is slight (Fig. 4). Thatis, a lower concentration of CCCP causes thesame degree of stimulation in partially induced

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175

150

E

+0

11000~~~~~~~~

z0

0 2 4 6 8 10[ONPG] (mM)

FIG. 3. ONPG transport in glucose-grown strainML308 as a function of ONPG concentration. Sym-bols: +, effect of ONPG concentration on transport incontrol cells; 0, non-energy-depleted cells treatedwith 20 MM CCCP at each concentration of ONPG; *,energy-depleted cells treated with 20 MAM CCCP ateach concentration of ONPG; E, ONPG transport byenergy-depleted cells. Each data point has a separateformaldehyde control and represents the net initialtransport rate with CCCP and ONPG added simulta-neously to initiate the reaction.

TABLE 2. Effect ofCCCP and FCCP on ONPGtransport in E. coli ML35

In vivo ONPGCellSa Assay condition hydrolysisCeliso Assay ~~~(Mmol/g

per min)

Non-energy No addition 9.1depleted 5 AM CCCP 9.5

1OAM CCCP 10.120 AM CCCP 10.25 uM FCCP 11.110AM FCCP 11.220MM FCCP 10.9

Energy depleted No addition 40.25AM CCCP 40.310OUM CCCP 41.020OM CCCP 40.95MM FCCP 40.410MM FCCP 42.120AM FCCP 42.8

a Condition: 0.2% Glucose-grown cells, when energydepleted, were treated for 60 min at 37 C.

cells than with cells that have maximal levels ofpermease.

Effect of other uncouplers. Proton conduc-tor types of uncouplers other than CCCP andFCCP were used in experiments similar to thosedescribed above. The results of these on strain

ML308 were mixed (data not shown). Tetra-chlorosalicylanilide stimulated transport in en-ergy-depleted cells to a degree similar to that forCCCP and FCCP, whereas pentachlorophenoland tetrachlorotrifluorobenzimidazole had littleeffect on energy-depleted cells. In addition,three independent workers in this laboratoryhave confirmed that azide does not stimulatedownhill transport in energy-depleted cells. Ev-idence has been presented (15) that azide actsas a proton conductor in addition to inhibitingthe terminal oxidase. The failure of these latteragents probably means that sufficient concen-tration in the membrane to support the neces-sary proton current was not achieved.The effect of energy depletion and of uncou-

plers on an adenosine 5'-triphosphatase mutantdescribed by Rosen (17, 18) was also investi-gated. This mutant is normal in having amembrane neither more nor less permeable toneutral polar substances than that of the paren-tal strain. This was shown by Rosen, and we canconfirm it because the rates of hydrolysis of ourformaldehyde controls were not higher than forthe cells of the parental strain. Rosen (18)presents some data to suggest that this mutantis specifically permeable to protons. We cannotsupport that contention because the downhilltransport of the mutant strain (Table 3) wasalmost as effectively inhibited by the energydepletion as were the parental strain (compare5.4 and 0.4 Mmol/g per min). Both parent strain7 and the adenosine 5'-triphosphatase strainNR70 are similarly stimulated by CCCP. If themutant's lesion were equivalent to the addition

100o I *

'75 00E

iii 50-

0

Y25

0 5 10 15 20[CCCP] (NLM)

FIG. 4. ONPG transport in glucose-grown strainML30 treated with CCCP. Strain ML30 was inducedfor 15 min with isopropyl-thio-j3-D-galactopyranosideand then treated as before for energy depletion. Sym-bols: 0, non-energy-depleted cells treated with CCCP.In each case, the subsequent formaldehyde controlwas subtracted for each data point. As before, ONPGand CCCP were added simultaneously to initiate thereaction. The data points represent the initial trans-port rates.

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TABLE 3. Effect of CCCP on ONPG transport instrains 7 and NR 70

Net in vivoONPG

Cellsa Assay conditions hydrolysis(Mmol/gper min)

A. Strain 7b No addition 137.7Non-energy 10MUM CCCP 110.0

depleted 20MM CCCP 74.7Energy depleted No addition 0.4

10 MmCCCP 31.020AM CCCP 25.9

B. NR 70C No addition 109.4Non-energy 10AM CCCP 80.0

depleted 20gM CCCP 81.9Energy depleted No addition 5.4

10 AM CCCP 25.120uM CCCP 33.4

a Conditions: Cells were grown on 2% glucose with0.5 mM isopropyl-thio-#-D-galactopyranoside and en-ergy depleted for 60 min.

b Parental strain.c Adenosine 5'-triphosphatase mutant.

of CCCP, then the addition of CCCP to the mu-tant should have had no effect. Further studieswith a variety of adenosine 5'-triphosphataseand energy-uncoupled mutants is indicatedsince the properties of the various mutants differconsiderably. A number of interpretationsmight be offered for the discrepancy betweenour results and Rosen's on the proton permea-bility of NR70. For example, NR70 may beselectively more permeable to SCN - necessaryfor the pulse-pH type of experiment.

DISCUSSIONThe results reported in this communication

indicate that thermodynamically downhilltransport is sensitive to the action of uncouplersclassified as proton conductors. ONPG trans-port under our experimental conditions is al-ways thermodynamically downhill, since theexcess of ,B-galactosidase inside the cell cleavesONPG as rapidly as it enters. This process isonly partially inhibited (at least initially) bylarge concentrations of proton conductors innon-energy-depleted cells. These are concentra-tions of CCCP that instantly block uptake orinitiate net efflux of radioactive non-utilizablethiogalactosides (16) such as thio-methyl-fl-D-galactopyranoside. However, these concentra-tions still do not block completely cellularhydrolysis of ONPG. Conversely, energy-de-pleted cells are stimulated in the presence ofCCCP and FCCP. Figures 5 and 6 give a

diagramatic representation of a possible expla-nation for these results. Figure 5 shows threedistinct transmembrane processes according toMitchell's (15) chemiosmotic hypothesis. Theprocess illustrated in the middle section of Fig.5 carries out the work of charge separationwhere 2 mol of protons are assumed to be movedacross the membrane without bringing theircorresponding anions. (We have representedthis as a partially blackened box.) This transfermay happen in conjunction with the hydrolysisof 1 mol of adenosine 5'-triphosphate or themovement of a pair of electrons past specificpoints in the electron transfer chain. For ourpurposes this flux can be represented as unidi-rectional. This transfer results in a membrane

CHEMIOSMOTIC MECHANISM

OUTSIDE

Y-GAL

H+

H+4

MEMBRANESYM SYMY-GdAL _YGASYM ,- SYM

IH+ H

CON- -CCON

HCON HCON

INSIDE

Y-GAL <

H+ GALHX

4H+

FIG. 5. Postulated systems in the membrane.SYM, Symporter; Y-GAL, any galactoside; HCON,proton conductor in unionized form; HX, unionizedspecies (such as water split by energy-requiring mem-brane-bound mechanism) (indicated by the blackenedarea in the diagram). This process and the hydrolysisof Y-GAL are the only ones assumed to be unidirec-tional.

NET FLUX PATTERNS

FIG. 6. Net flux of protons and galactosides in thepresence or absence of proton conductors and/orenergy metabolism. In all cases Y-GAL (any galacto-side) is maintained low inside the cell by the action of,8-galactosidase. The relative rates of hydrolysis in thefour cases is indicated by the size of the lettering.

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potential which, because of the thinness of thecytoplasmic membrane, is typically in the rangeof 105 V/cm (see Appendix). In spite of theassumption that hydrogen ions are moved,under most conditions (ours included) thebuffer capacity is so high, both inside andoutside, that there are no significant pHchanges resulting from the extrusion, and move-ment of charged species present in the mem-brane are essentially the electrophoretic onesdue to the high-voltage gradient. Second, themodel further proposes that charge (specificallyin the form of protons) gives the symporter forgalactosides a net charge but can bind appropri-ately only if a galactoside is also bound. Thecharge can retraverse the membrane throughthe 3-galactoside-proton-symporter which, inci-dentally from the point of view of the proton,drives the entry of a galactoside molecule(upper part of Fig. 5). Third (bottom part ofFig. 5), the presence of a proton conductor suchas CCCP or FCCP (symbolized by HCON)allows the migration of charged protons in thedirection required by the ambient electricalfield. When present these agents should uncou-ple the system by permitting re-exit of protonsunlinked to galactoside transport of protonsback across the membrane and down theirelectrochemical gradient, collapsing the chargeseparation and the potential. Under these cir-cumstances, any 0-galactoside would be free tocross the membrane but would not be aided by ametabolic protonmotive force. Transport wouldstill take place regardless of the previous energystatus of the cell because no protonmotive force,either favorable as developed by cellular me-tabolism or unfavorable as developed by thedownhill symport process itself, could be sus-tained (see both lower panels of Fig. 6). In thespecific case of a hydrolyzable galactoside suchas ONPG, entry would continue indefinitely,since cleavage of the molecule internally wouldprevent equilibration of ONPG on both sides ofthe membrane.

Since the energy-depleted cells (Fig. 6 upperright-hand panel) have been made to utilizetheir energy reserves, they are not capable ofextruding protons coupled to metabolism.Thus, no membrane potential (or pH differ-ence) is maintained across the membrane andno active transport can take place. Initially,ONPG can enter via the permease-mediatedpathway, carrying protons along with it. How-ever, it can be calculated that when 0.12 Amol ofONPG/g (dry weight) enters the cell, a reversepotential of 90 MV will develop within the orderof 25 to 50 ms, preventing or stalling further netmovement (see Appendix). However, when an

uncoupler like CCCP is added (Fig. 6, lowerright-hand panel), allowing the reverse proton-motive force to be discharged, ONPG moleculescan now enter on the symporter without thesystem becoming stalled. The symporter-protonconductor combination can simultaneouslycarry ONPG and a proton down their respectiveelectrochemical gradients, although the sym-porter also carries a proton against its gradient.Of course, the total gradient must be in thedirection of net flux. In essence, the uncouplerallows a quasi-facilitated diffusion system morecomplex than that usually considered becausethere are two carriers: the permease and theproton conductor. In non-energy-depleted cells,the uncoupler short-circuits the active extrusionof protons, and thus only this same rate ofquasi-facilitated diffusion occurs.

This interpretation is supported by our re-sults (Table 2) in that transport-negativestrains such as ML35 are not stimulated byuncouplers. Thus, a functional permease isrequired for transport in the presence of uncou-plers.The recent report of Kaback et al. (11) that

CCCP acts as a sulfhydryl reagent in membranevesicles is also of interest. In the case of fl-galac-toside transport, since it is known that the Mprotein has a sensitive sulfhydryl residue (3), itwould not be unexpected that CCCP wouldprogressively inactivate the permease as well asshort-circuit proton currents. Both processes dotake place. The former seems to have a half-time of about 20 min with 20,uM CCCP, and thelatter appears to be fully established within 1min. Thus, the experiments reported herewith CCCP were essentially completed beforethe inactivation became marked. Therefore, wewould suggest that increased proton conductiv-ity induced by CCCP supersedes the sulfhydryleffect of the compound. FCCP and tetra-chlorosalicylanilide also stimulate transport inenergy-depleted cells similar to that seen withCCCP, and these compounds have not beenshown to be sulfhydryl reagents although it maynot be unlikely that FCCP would behave simi-larly to CCCP in this respect. In addition,reducing agents such as dithiothreitol do notreactivate energy-depleted cells (14). Togetherthese observations suggest that any effect on thesulhydryl group of the permease is secondary tothe major effect reported here.

It would be expected that an ionophore suchas valinomycin may allow K+ efflux to compen-sate for proton entry (19) and should thus allowat least a limited ONPG hydrolysis to takeplace. However, treatment of energy-depletedcells with ethylenediaminetetraacetic acid to

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allow valinomycin to interact with the cytoplas-mic membrane renders the energy-depletedcells extremely leaky (data not shown), makingsuch experiments unsuccessful in these cells.

It is appropriate to comment on the lack ofstimulation of downhill transport energy-de-pleted cells of all substances classified as protonconductors. Clearly, the substances so classifiedvary widely in their proton-conducting proper-ties, no doubt depending on their pK values (5,8) and the degree of delocalization of the chargein their ionized forms.

It appears likely that effective proton conduc-tion is necessary to get this artificial two-carri-er-facilitated diffusion system to function. Itshould be pointed out that none of these mole-cules undergoes the kind of conformationalchange that valinomycin undergoes when itbinds potassium and completely surrounds thecharge with nonpolar groups. Consequently, itcan be argued that none of the artificial protonconductors is, in fact, highly effective.

In toto, these results are in agreement withthe ideas proposed by Mitchell (15, 19) and, inour view, provide strong critical evidence forthem. However, they do not seem compatiblewith the previous ideas of Kaback and co-work-ers (9, 10), since it is not obvious how protonconductors would stimulate transport accordingto the model of direct coupling of electron flowto transport.

APPENDIXStorage capacity of the bacterial mem-

brane for electrical charge. Typical restingpotentials for biological membranes are near- 80 to -160 mV. If the thickness of the insulat-ing bilayer is 8 nm, then the voltage gradient is 1x 105 to 2 x 105 V/cm. Consequently, chargedspecies mobile in the membrane are subject tostrong electrophoretic forces. Two- or threefold-larger potentials result in membrane rupture.On the other hand, the capacity of the mem-

brane to store charge is very limited, so thattransport via the chemiosmotic or co-transportmechanisms require on-going active rechargingof the membrane for transport to continue.To calculate the charge storage capacity of

the bacterial cell membrane, we assume thatthe cell membrane is a spherical shell of radius(r) 8 x 10-5 cm, of membrane thickness (b) 8 x10-7 cm, and of dielectric constant (e) 6, typi-cal of phospholipids. For shell geometry, if thecapacitance (C) is given in pF and the dimen-sions are in centimeters,

C - 1.112 e r2/6 = (1.112) (6) (8 x10-5) 2/(8 x 10)-7 = 53.37 x 10-3 pF

For an assumed voltage (V) of -0.090 volts,since CV - Q; the charge (Q) is given by:Q = 0.090 x 53.37 x 10-15

= 4.80 x 10-15 coulomb/cellThis can be converted to moles of charge pergram (dry weight) of cells with the use of theFaraday (96,501 coulombs/mol) and the dryweight of a cell, which is roughtly4/3 (11 r3 p) = 4/3 x 3.14 x (8 x 10-5)s x 0.2

= 4.29 x 10-13 g/cell

assuming that p, the density of dry solids, is 20%of the wet weight.

This leads to a storage capacity of:

4.80 x 10-15/(4.29 x 10-13 x 96,501)= 1.16 x 10-7 mol/g (dry weight)

This value of 0.116 umol/g (dry weight) is tobe compared with the steady-state rate ofhydrolysis of ONPG by non-energy-depletedcells of 150 to 250 ,mol/g (dry weight) per min.Consequently, it would take from 46 to 28 ms atsuch rates to develop a 90-mV reverse potentialwhen energy-depleted cells are supplied withONPG if transport continued undiminished un-til stalled.This order of magnitude suffices to show that

the maximum energy stored in electrical capaci-tance at the highest protonmotive forces imagi-nable would support less than a second's worthof transport at typical steady-state rates.

ACKNOWLEDGMENTS

Work in our laboratory is supported by National ScienceFoundation grant GB-32115 and by Public Health Servicegrant AI-09337 from the National Institute of Allergy andInfectious Diseases.We thank Dale Pardy for communicating to us the re-

sults of his unpublished experiments.

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