Vol. 170, No. 9JOURNAL OF BACTERIOLOGY, Sept. 1988, p. 4330-43370021-9193/88/094330-08$02.00/0Copyright © 1988, American Society for Microbiology

Sensitivity of Some Marine Bacteria, a Moderate Halophile, andEscherichia coli to Uncouplers at Alkaline pH

ROBERT A. MAcLEOD,* G. A. WISSE, AND F. L. STEJSKALDepartment of Microbiology, Macdonald College of McGill University, 21,111 Lakeshore Road,

Ste Anne de Bellevue, Quebec, Canada H9X JCOReceived 23 February 1988/Accepted 27 May 1988

The inhibitory effects of uncouplers on amino acid transport into three marine bacteria, Vibrio alginolyticus118, Vibrio parahaemolyticus 113, and Alteromonas haloplanktis 214, into a moderate halophile, Vibrio costicolaNRC 37001, and into Escherichia coli K-12 were found to vary depending upon the uncoupler tested, itsconcentration, and the pH. Higher concentrations of all of the uncouplers were required to inhibit transportat pH 8.5 than at pH 7.0. The protonophore carbonyl cyanide m-chlorophenylhydrazone showed the greatestreduction in inhibitory capacity as the pH was increased, carbonyl cyanide p-trifluoromethoxyphenylhydrazoneshowed less reduction, and 3,3',4',5-tetrachlorosalicylanilide was almost as effective as an inhibitor of aminoacid transport at pH 8.5 as at pH 7.0 for all of the organisms except A. haloplanktis 214. Differences betweenthe protonophores in their relative activities at pHs 7.0 and 8.5 were attributed to differences in their pK values.3,3',4',5-Tetrachlorosalicylanilide, carbonyl cyanide m-chlorophenylhydrazone, 2-heptyl-4-hydroxyquinoline-N-oxide, and NaCN all inhibited Na+ extrusion from Na+-loaded cells of V. alginolyticus 118 at pH 8.5. Theresults support the conclusion that Na+ extrusion from this organism at pH 8.5 occurs as a result of Na+/H+antiport activity. Data are presented indicating the presence in V. alginolyticus 118 of an NADH oxidase whichis stimulated by Na+ at pH 8.5.

In 1981, Tokuda and Unemoto (32) showed that themembrane potential (At) of K+-depleted and Na+-loadedVibrio alginolyticus 138-2 was completely collapsed by 10,uM (4 nmol/mg of cell dry weight [in all references toconcentration in this paper, the term in parentheses refers tothe number of nanomoles or micromoles of the compoundtested per milligram of cell dry weight]) carbonyl cyanidem-chlorophenylhydrazone (CCCP) at pH 6.0 to pH 7.0 butwas only partially and transiently collapsed by this uncou-pler at the same concentration at pH 8.5. They also foundthat respiration-dependent Na+ extrusion from the cellswhich occurred in the presence of K+ as well as Na+-dependent uptake of a-aminoisobutyric acid (AIB) wasinhibited by 10 ,uM (2.2 nmol/mg) CCCP completely at pH6.5 but only slightly at pH 8.5. To explain these observationsthe authors proposed the existence of a primary electrogenicNa+ extrusion system (Na+ pump) in V. alginolyticus 138-2which they later concluded functioned at the Na+-activatedNADH:quinone-acceptor oxidoreductase segment of therespiratory chain (35). Further evidence for the existence ofan Na+ pump was obtained when mutants of V. alginolyticus138-2 were isolated which showed (i) increased sensitivity toCCCP during growth, (ii) an Na+ extrusion mechanismwhich was sensitive to 10 ,uM (2.9 nmol/mg) CCCP, and (iii)NADH oxidase activity, no portion of which was stimulatedby Na+ (28).Tsuchiya and Shinoda (36) observed respiration-driven

Na+ extrusion from Vibrio parahaemolyticus at pH 8.5which was insensitive to 20 puM (3 nmol/mg) CCCP. Ken-Dror et al. (16, 17) found that at pH 8.5, 5 puM (0.07 nmol/mg) carbonyl cyanide p-trifluoromethoxyphenylhydrazone(FCCP) slightly increased the rate of Na+ extrusion fromNa+-loaded cells of the halotolerant bacterium Ba1. Theyconcluded that a respiration-linked, uncoupler-insensitiveNa+ pump was responsible for the effect observed. Dibrov

* Corresponding author.

et al. (4) examined the effects of Na+ and inhibitors onNADH oxidation and AT formation by inverted cytoplasmicmembrane vesicles of V. alginolyticus and concluded thattheir results were in agreement with the suggestion ofTokuda and Unemoto that a primary electrogenic Na+ pumpexists in the membrane of this bacterium. From theseobservations it was concluded that moderately halophilicbacteria demonstrate two different types of energetics de-pending on the external pH. At an acidic pH, only the protonmotive force is generated as the immediate result of respira-tion, whereas at an alkaline pH, the generation of the Na+electrochemical potential is a primary process performed bythe Na+ pump (25, 34).

In view of the reports that moderate halophiles becomeuncoupler resistant at an alkaline pH, we were interested tofind that amino acid transport into V. alginolyticus 118 andsome other moderate halophiles was inhibited by and wasalmost as sensitive at pH 8.5 as at pH 7 to the protonophore3,3',4',5-tetrachlorosalicylanilide (TCS). Further investiga-tion revealed that V. alginolyticus 118 also possesses an Na+extrusion mechanism which is sensitive to protonophores atan alkaline pH.

MATERIALS AND METHODSOrganisms. V. alginolyticus 118 and V. parahaemolyticus

113 were obtained from P. Baumann (University of Califor-nia, Davis), Vibrio costicola NRC 37001 was from D.Kushner (University of Ottawa, Ottawa, Ontario, Canada),Escherichia coli K-12 (wild type [27]) was from the E. coliGenetic Stock Center, Yale University, New Haven, Conn.,and Alteromonas haloplanktis 214 ATCC 19855 was fromour laboratory.Growth and harvesting of cells. For transport studies, cells

of the marine bacteria and the moderate halophile weregrown on a rotary shaker to the stationary phase in acomplex medium containing 0.8% nutrient broth (DifcoLaboratories, Detroit, Mich.), 0.5% yeast extract (Difco), 50

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mM MgSO4, 10 mM KCl, and concentrations of NaCl whichvaried with the organism. The NaCl concentrations andgrowth temperatures, respectively, were as follows: for V.alginolyticus 118, 400 mM and 25°C; for V. parahaemoly-ticus, 200 mM and 37°C; for V. costicola, 1 M and 30°C; andfor A. haloplanktis, 300 mM and 25°C. E. coli was grown inL broth (21) at 30°C. Cells were harvested by centrifugation(10,000 x g for 10 min at room temperature) and washedtwice with either potassium 3-(N-morpholino)propane-sulfonate (MOPS) buffer (pH 7.0) or N-tris(hydroxymethyl)methylglycine (Tricine) buffer (pH 8.5) at room temperature.The buffers contained 50 mM MOPS or Tricine together with10 mM MgCl2, 10 mM KCI, and NaCl at the concentrationpresent in the growth medium. The same buffers were usedto wash E. coli, except that 100 mM NaCI was added.For At and Na+ extrusion studies, V. alginolyticus was

grown in the synthetic medium of Tokuda et al. (30) to theearly log phase. To obtain enough log-phase cells for anexperiment, a sufficient quantity of an overnight culture ofthe organism grown in the synthetic medium was added tofresh synthetic medium to bring the optical density at 660 nmof the suspension to 0.1 to 0.12. The volume of mediuminoculated depended on the yield of cells required. Theinoculated medium was incubated on a rotary shaker at 25°Cuntil the optical density at 660 nm had increased to 0.2 to0.25 (after 2 to 3 h). Cells from such a culture werepenetrable by tetraphenylphosphonium ion (TPP+) and di-ethanolamine; cells from stationary-phase cultures were not.Cells for At studies were harvested and washed twice ineither 50 mM MOPS buffer (pH 7.0) or 50 mM Tricine buffer(pH 8.5) each containing 400 mM NaCl but no KCl or MgCl2.Cells for Na+ extrusion experiments were harvested andwashed as described below.

Estimate of cell dry weight and cell protein. The dry weightof cells was estimated spectrophotometrically by assumingthat an E660 of 1.0 was equivalent to 0.385 mg of cell dryweight per ml (as determined for A. haloplanktis 214 [5]).Cell protein was determined by the biuret procedure (11).For V. alginolyticus 118, an E660 of 1.0 was found to beequivalent to 0.279 mg of cell protein per ml.

Inhibitor concentrations reported in other publicationshave been expressed here in terms of cell dry weight. Sincein some publications cell concentrations have been reportedas cell protein, we have estimated cell dry weight byassuming the dry weight to be equivalent to 66% protein (11).

Transport studies. The procedures used have been de-scribed previously (5), except for the following modifica-tions. The incubation medium contained the same salts at thesame concentrations as the washing buffer. The cell concen-tration used was the equivalent of 100 pug of cell dry weightper ml. Inhibitors were added to the cell suspensions 15 minprior to the addition of the exogenous energy source, whichwas glycerol at 25 mM for all of the organisms except A.haloplanktis. For the latter, ethanol at 25 mM was provided.The reaction was started by adding either L-1-'4C-AIB or D-[U-14C]alanine at 200 puM (0.5 p.CiIp.mol). Samples wereremoved at 30-s intervals during the first 3 min of uptake andfiltered through 0.45-pum-pore HA filters (Millipore Corp.,Bedford, Mass.). The cells retained on the filters werewashed immediately with 5 ml of the washing buffer. Radio-activity was measured with a scintillation counter. Initialrates of uptake were determined based on the uptake ofradioactivity during the first minute.

Respiration measurements. The procedures used havebeen described previously (18). The incubation mediumcontained the same salts at the same concentrations as were

used in the transport studies. The cell suspensions, with andwithout inhibitors, were incubated with aeration for 15 minprior to being added to the reaction chamber of the oxygenelectrode. After the rate of endogenous oxygen uptake wasdetermined, the rate in the presence of the exogenouselectron donor was determined. The cell concentration usedwas the equivalent of 333 ,ug of cell dry weight per ml.At measurements. The equilibrium distribution of [3H]

TPP+ was measured by using slight modifications of theprocedure of Tokuda and Unemoto (33). Cells to provide 2mg of cell protein per ml were incubated in a series of testtubes (10 by 75 mm) each containing 50-pI volumes of either50 mM MOPS buffer (pH 7.0) or 50 mM Tricine buffer (pH8.5) containing 400 mM NaCl and 20 mM glycerol. Inhibitorswere added as solutions in methanol (H20 for KCN) in afixed volume of 1 pI 15 min prior to the start of theexperiment. An equal volume of methanol or H20 was addedto control tubes. Assays were started by adding [phenyl-3H1tetraphenylphosphonium bromide (New England NuclearCorp., Boston, Mass.) at 19 ,uM (157 ,uCi/,umol). Afterincubation for 15 min at 25°C, uptake was terminated byadding 2 ml of 0.4 M NaCI, filtering the mixtures throughGA-6 cellulose acetate membrane filters (Gelman Sciences,Inc., Ann Arbor, Mich.), and washing the filters with 2 ml of0.4 M NaCl. Radioactivity was measured with a scintillationcounter. Identical incubation mixtures containing no addedcells were filtered through membrane filters to correct forradioactivity bound to the filters. Calculations of AT weremade by using an intracellular fluid volume for V. alginoly-ticus of 3.3 RI/mg of cell protein (32) and applying the Nernstequation:

= -59 log [TPP+] in

[TPP+] out

Measurements of Na+ extrusion. Cells of V. alginolyticus118 were loaded with Na+ by a modification of the procedureof Tokuda (29). Log-phase cells of V. alginolyticus 118grown on synthetic medium were harvested by centrifuga-tion (10,000 x g for 10 min at 25°C) and suspended at 25°Cfor 10 min in a volume of 50 mM diethanolamine hydrochlo-ride (pH 8.5)-0.4 M NaCl equal to one-half the volume of thegrowth medium. The cells were suspended a second time ina solution of the same composition and volume and incu-bated at 25°C for 1 h. The cells were collected by centrifu-gation and washed twice by suspension in and centrifugationfrom a volume of 50 mM Tricine-NaOH (pH 8.5)-0.4 MNaCl also equal to one-half the volume of the growthmedium. The washed cells were suspended in 0.4 M NaCl ata cell density equivalent to 50 mg of protein per ml andmaintained on ice. A sample of this suspension was dilutedwith a sufficient quantity of 50 mM Tricine-NaOH (pH 8.5)-0.4 M NaCl to yield a cell density equivalent to 2 mg ofprotein per ml. 22Na' as carrier-free NaCl (AmershamCanada Ltd., Oakville, Ontario, Canada) was added to yield104 cpm/,l, and the cells were stored on ice in a cold roomat 4°C for 18 to 22 h.

Aliquots (50 RI) of the cell suspension were transferred toa series of test tubes (10 by 75 mm) maintained on ice. Foreach of the conditions tested, tubes were set up in triplicate.Inhibitor solutions (in H20 for NaCN and in methanol for2-heptyl-4-hydroxyquinoline-N-oxide [HOQNO], TCS, andCCCP) were added in 1-pA volumes and incubated with thecells for 15 min in the cold. Controls containing 1-pA volumesof H20 only and methanol only were included. Immediatelyprior to the start of the experiment 2 RI of a solutioncontaining KCI and glycerol at concentrations designed to

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4332 MAcLEOD ET AL.

(a) V. alainolyticus 1 18CCCP FCCP TCS

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PROTONOPHRO CONCENRATION (PM)

Curves 1 pH 7.0Curves 2 pH 8.5

FIG. 1. Capacity of the protonophores CCCP, FCCP, and TCS to inhibit the initial rate of uptake of AIB into V. alginolyticus, V.parahaemolyticus, and V. costicola and of D-alanine into A. haloplanktis and E. coli at pHs 7.0 and 8.5. Initial rates of uptake in the absenceof inhibitors at pHs 7.0 and 8.5 were 42 and 53 (a), 8 and 15 (b), 50 and 54 (c), 23 and 35 (d), and 10 and 15 (e) nmol/mg of cell dry weightper min. The cell concentration in the incubation mixtures was 100 ,ug (dry weight) per ml. In these experiments, a 1 ,uM concentrationrepresented 10 nmol of inhibitor per mg of cell dry weight.

yield final concentrations of 10 mM KCl and 25 mM glycerolwas added to each suspension. To start the experiment thetubes on ice were placed in a water bath shaker at 25°C. Ateach time interval, 2 ml of 0.4M NaCl was added to each cellsuspension, and the suspension was filtered immediatelythrough polysulfone filters (GA6-S, 0.45-,um pores; Gelman)and washed once with 2 ml of 0.4 M NaCl. The filters weredried under an infrared lamp and placed in scintillation vials,a scintillator (MP Ready-Solv; Beckman Instruments, Inc.,Palo Alto, Calif.) was added, and radioactivity was mea-sured in a liquid scintillation counter (LS 7500; Beckman) byusing the 32p program.K+ determination. For determination of intracellular K+,

cells equivalent to a known amount of protein per milliliterwere suspended in 0.1 M HCl, heated to 100°C for 10 min,and centrifuged from the suspension. The concentration ofK+ in the supematant solution was determined by flamephotometry with an SP9 series atomic absorption spectrophotometer (Pye Unicam Ltd., Cambridge, England) oper-ating in the flame photometer mode. Intracellular K+ con-centrations were calculated based on an intracellular fluidvolume for V. alginolyticus of 3.3 ,lp/mg of cell protein (31).

RESULTS

Effects on transport. The effects of three different proton-ophores on amino acid uptake by three Na+-requiring ma-rine bacteria, V. alginolyticus, V. parahaemolyticus, and A.haloplanktis, an Na+-dependent moderate halophile, V.costicola, and a non-Na+-dependent species, E. coli, wereexamined (Fig. la to e). For the three marine bacteria andthe moderate halophile, all of the uncouplers tested wereeffective in inhibiting either AIB or D-alanine transportcompletely at pH 7.0 at concentrations of 1 to 5 ,M (10 to 50nmol/mg). For E. coli at pH 7.0, concentrations required forcomplete inhibition of transport were appreciably higher, 25to 50 ,uM (250 to 500 nmol/mg). At pH 8.5 the extent of the

inhibitory effect varied with the protonophore and the or-ganism. For all of the organisms CCCP was less effective atpH 8.5 than at pH 7.0 in inhibiting amino acid transport atlow concentrations but was still 80 to 100% effective at ahigher concentration (50 ,uM) (0.5 ,umollmg). With the ex-ception of the moderate halophile, V. costicola, the differ-ences in the effects of CCCP at pHs 7.0 and 8.5 were greaterfor the Na+-requiring species than for non-Na+-requiring E.coli. TCS, however, with one exception, inhibited transportof the organisms completely at both pHs, and inhibition atlow concentrations was only slightly lower at pH 8.5 than atpH 7.0. For instance, for V. alginolyticus 118, 1 ,uM (10nmol/mg) TCS was inhibitory at pH 7.0, and 3 ,uM (30 nmollmg) TCS was inhibitory at pH 8.5. For A. haloplanktis, TCSwas much less inhibitory for transport at pH 8.5 than at pH7.0 at low concentrations, although at 50 ,uM (0.5 ,umol/mg)at pH 8.5, TCS inhibited transport by more than 80%. For allof the organisms tested, FCCP produced effects intermediatebetween those of CCCP and TCS.

Effects on respiration. Since with one exception pH hadvery little effect on the sensitivity of the organisms tested toTCS, the possibility was considered that the mechanism ofinhibition of transport by this uncoupler might be differentand relatable to effects on respiration. Table 1 shows that allthree protonophores inhibited the respiration of V. alginoly-ticus to a small extent at pH 8.5, but their effects onrespiration did not parallel their effects on transport. Asubsequent experiment with V. alginolyticus at pH 8.5confirmed the slight effect ofTCS on respiration at 10 ,uM (30nmollmg) but showed that at 50 ,uM (150 nmollmg) theprotonophore inhibited respiration 86% (data not shown).

Effects of TCS and other inhibitors on At. The effects ofTCS concentration on the AT of V. alginolyticus 118 wereexamined by using [3H]TPP+ as a probe (Table 2). At pH 7.0as little as 5 p,M (1.65 nmol/mg) TCS reduced At to 0, whileat pH 8.5 5 ,uM (1.65 nmol/mg) TCS reduced Al' 32% and 20

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TABLE 1. Effect of protonophores on the rate of respiration ofV. alginolyticus 118 at pH 8.5

Protonophore Rate of Inhibition ofaddeda respiration' respiration (%)

None 241 0CCCP 188 22.0FCCP 151 37.3TCS 165 31.5

a At 10 FM (30 nmol/mg of cell dry weight).b Reported as nanogram atoms of oxygen per milligram of cell dry weight

per minute.

,uM (6.60 nmol/mg) TCS reduced it to 0. In the absence ofTCS, At was appreciably higher at pH 8.5 than at pH 7.0, ashas also been observed with V. costicola (7).

In the course of these studies it was noted that there wasa TCS-dependent binding of [3H]TPP+ to the celluloseacetate membrane filters which increased as the concentra-tions of TCS in the incubation mixture increased. Correc-tions had to be made for this very considerable binding toenable calculations of AtI to be made. CCCP did not cause[3H]TPP+ to be bound to the filters (results not shown).Tests with butanol-treated cells (13) showed that atiy errorswhich might be due to [3H]TPP+ bound to the cells fellwithin the litnits of error of the methods used to measure

The uncoupler-insensitive accumulation of [3H]TPP+ intomarine bacteria and moderate halophiles has been ascribedto the operation in these organisms of an electrogenic Na+pump (32-35). At an uncoupler concentration (20 pLM) (3.1nmol/mg) which did not reduce the At of V. costicolacompletely, a further addition of the respiratory inhibitorNaCN at 10 mM (9.1 ,umol/mg) reduced the AT almost to 0,leading the authors to conclude that the pump is energydependent (37). At pH 8.5 a combination of 10 mM (3.3p.mollmg) KCN and 5 p.M (1.65 nmol/mg) TCS or of 50 ,uM(16.5 nmol/mg) HOQNO and 5 ,uM (1.65 nmollmg) TCSreduced the TCS-insensitive AtI of V. alginolyticus 118 to 0(Table 3). The inhibitors alone, however, did not have thiseffect.

Effect of protonophores on Na+ extrusion. Tokuda andUnemoto (32, 33) showed that Na+-loaded cells of V.alginolyticus 138-2 extruded Na+ if K+ was added to thesuspending medium. If K+ was omitted, extrusion wasprevented completely. In the presence of K+, Na+ extrusionby V. alginolyticus 138-2 was inhibited by 10 ,uM (2 nmol/mg) CCCP at pH 6.5 but not at pH 8.5. In our studies therates of extrusion of Na+ from Na+-loaded cells of V.alginolyticus 118 were very rapid in the presence of K+; inthe absence of added K+, these rates were reduced to

TABLE 2. Effect of TCS concentration on the APY ofV. alginolyticus 118 at pHs 7.0 and 8.5

ATb at pH:TCSa

7.0 8.5

0 -166 (+3.6%) -189 (±6%)5 (1.65) 0 -128 (+20%)10 (3.30) 0 -120 (±17%)20 (6.60) 0 0a Values represent the micromolar concentration (nanomoles of TCS per

milligram of cell dry weight).b Values represent the average (average deviation) of triplicate determina-

tions.

TABLE 3. Effect of respiratory inhibitors on the 'Vl ofV. alginolyticus 118 at pHs 7.0 and 8.5 in the presence and

absence of TCS

A^'I at pH:Inhibitor(s) addeda

7.0 8.5

None -167 (±3.6%) -172 (+7%)TCS (5 ,M) 0 -137 (±5%)KCN (10 mM) -102 (±7%) -186 (±11%)KCN (10 mM) + TCS (5 ,uM) _c 0HOQNO (50 ,uM) -100 (+9%) -153 (+12%)HOQNO (50 ,uM) + TCS (5 ,uM) - 0

a In this experiment, inhibitor concentrations of 5 and 50 ,uM represent 1.65and 16.5 nmol, respectively, and that of 10 mM represents 16.5 ,umol (per mgof cell dry weight).

I Values represent the average (average deviation) of triplicate determina-tions. Experiments at pHs 7.0 and 8.5 were carried out on different batches ofcells.c-, Not tested.

extents which varied from experiment to experiment. In oneexperiment, analysis of the supernatant of an incubationmixture without added K+ after separation of the cells bycentrifugation revealed that K+ was present as a contami-nant at a concentration of 30 ,uM.To demonstrate the effects of inhibitors on Na+ extrusion

by V. alginolyticus 118, we introduced modifications to theprocedures described by Tokuda and Unemoto (31, 32) andTokuda (28) (see Materials and Methods). In the absence ofinhibitors, the initial rates of extrusion of Na+ against theinwardly directed Na+ gradient were very rapid, and extru-sion was complete in 1 to. 3 min, depending upon theexperiment. When 20 ,uM (6.6 nmol/mg) TCS was present inthe suspension (Fig. 2A), there was an initial drop in cellNa+, after which the retention of Na+ remained constant atabout 70%. Raising the concentration of TCS to 50 ,uM (16.5nmol/mg) caused an 85% retention of radioactivity (resultsnot shown). CCCP at 20 p.M (6.6 nmol/mg) reduced the rateof Na+ extrusion but did not stop it completely (Fig. 2A).The respiratory inhibitor HOQNO at 50 ,uM (17 nmol/mg) orNaCN at 20 mM (6.6 ,umol/mg) resulted in an initial loss of22Na+ from the cells, after which further extrusion of Na+ceased (Fig. 2B). The addition of iodoacetate at 20 mM (6.6,umol/mg) to inhibit glycolysis, together with CN-, failed toincrease the retention of Na+ over that produced by CN-alone or to reduce the point scatter of the curves obtained(results not shown). The response to NaCN tended to beerratic.

Effects of Na+ on NADH oxidation. Tokuda (29) hasisolated mutants of V. alginolyticus 138-2 which have anNa+ extrusion system which is inhibited by 10 ,uM (2.9 nmol/mg) CCCP at pH 8.5. Inverted membrane vesicles preparedfrom these mutants have NADH oxidase activities which arenot stimulated by Na+. Since the Na+ extrusion system ofV. alginolyticus 118 is also inhibited by protonophores at pH8.5, it was of interest to determine if the cells possess anNADH oxidase activity which is also not stimulated by Na+.To test for the oxidation ofNADH and succinate by their

respective intracellular enzymes, we made the cells perme-able by treating them with toluene by procedures which havebeen described previously (18). Such cells oxidized NADHin the absence of added Na+, and the rate at pH 8.5 was 43%faster than that at pH 7.0 (Table 4). The addition of Na+further stimulated NADH oxidation by 46% at pH 7.0 and by66% at pH 8.5. Succinate was also oxidized in the absence ofadded Na+, and again, rates of oxidation were faster at pH

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INCUBATION TIME(min)

FIG. 2. Effect of inhibitors on the extrusion of Na+ from Na+-loaded cells of V. alginolyticus 118 at pH 8.5. The highest radioac-tivity retained by the cells (in the presence of 20 ,uM TCS at timezero) was taken to be 100%. All counts were corrected for radioac-tivity bound to the filters. Each point recorded represents theaverage of triplicate determinations. The cell concentration in theincubation mixture was 3.1 mg (dry weight) per ml. Inhibitorconcentrations were as follows: TCS and CCCP, 20 ,uM (6.6 nmol/mg); HOQNO, 50 puM (17 nmol/mg); NaCN, 20 mM (6.6 p.mol/mg).

8.5 than at pH 7.0. In the presence of Na+, the rates ofsuccinate oxidation were reduced at both pHs. The experi-ments were repeated with intact cells as a control. The latteralso oxidized NADH at rates which were increased by Na+but which were slower than the rates With toluene-treatedcells and which were slower at pH 8.5 than at pH 7.0.Succinate was also oxidized by intact cells but at rates whichwere greatly increased at pH 7.0 (but only slightly at pH 8.5)by added Na+. As the values in parentheses show, endoge-nous rates were low in intact cells and negligible in toluene-treated cells. Results similar to those in Table 4 wereobtained in a second experiment.

DISCUSSIONMost of the marine bacteria and the mnoderate halophile

examined in this study have been shown previously to

TABLE 4. Effect of Na+ on the rate of oxidation of NADH andsuccinate by intact cells and toluene-treated cells of

V. alginolyticus 118 at pHs 7.0 and 8.5

Rate of oxidation" at the indicated pH in the

NaCla following cells:

Substrate a(MM) Toluene treated Intact

7.0 8.5 7.0 8.5

NADH 0 188 (8) 268 (0) 124 (10) 93 (5)NADH 400 274 (0) 446 (0) 191 (24) 123 (19)Succinate 0 44 (4) 139 (0) 35 (8) 82 (7)Succinate 400 13 (5) 63 (11) 124 (22) 95 (19)

a In the absence of NaCI osmotic balance was maintained by adding 400mM choline chloride.

b Reported as nanogram atoms of oxygen per minute per milligram of celldry weight. All values were corrected for endogenous oxygen uptake. Thenumbers in parentheses are the endogenous rates.

accumulate amino acids by Na+-dependent mechanisms,and there is evidence that uptake is energized by an electro-chemical potential of Na+ (20, 24, 31, 35). All have respira-tory systems which in inverted membrane vesicles or tolu-ene-treated cells are activated by Na+ (18, 39), and the siteof activation in V. alginolyticus 138-2 has been shown to beat the NADH:(quinone-acceptor) oxidoreductase step (38).E. coli, on the other hand, transports D-alanine by a proton-symport mechanism energized by an electrochemical poten-tial of protons (3) and has a respiratory system which is notactivated by Na+ (39).The results presented here indicate that membrane trans-

port into all of the organisms tested is sensitive to uncouplersat an alkaline pH provided the appropriate uncoupler ispresent at the right concentration. The most effective uncou-pler tested was TCS, and for most of the organisms it wasalmost as inhibitory at pH 8.5 as at pH 7.0. The only majorexception was A. haloplanktis, which showed a markedreduction in sensitivity to low concentrations of TCS at pH8.5. Even with this organism, inhibition of transport at pH8.5 was reduced 80% by 50 ,uM (0.5 ,umol/mg) TCS. Ourfindings with V. costicola confirm those of Hamaide et al.(8), who observed that 20 ,uM (40 nmollmg) TCS or CCCP atpH 8.8 inhibited AIB transport into this organism 99 and98%o, respectively.Our results with V. alginolyticus 118 showed that 20 ,uM

(6.6 nmol/mg) TCS at pH 8.5 reduced the AtI to 0. Hamaideet al. (8) found that 20 ,uM (40 nmollmg) TCS or CCCP at pH8.8 reduced the At of V. costicola 68 and 72%, respectively.These findings support the conclusion that the inhibition ofmembrane transport by TCS and CCCP at an alkaline pH inthese two organisms is due to the action of the compounds asprotonophores.

Unlike TCS, CCCP was much less effective in inhibitingmembrane transport at pH 8.5 than at pH 7.0, and thequestion arises as to why this is so. Tokuda and Unemoto(33) observed that the addition of 10 ,uM (4 nmol/mg) CCCPto a suspension of V. alginolyticus 138-2 at pH 8.5 collapsedAt only transiently but caused the intracellular pH todecrease. They concluded that since the cytoplasmic mem-brane was made permeable to protons by the CCCP, the APobserved at pH 8.5 was generated by a pump which extrudedan ion(s) other than H+. A protonophore, however, shouldbe able to collapse AP irrespective of whether it is generatedby the extrusion of Na+ or of H+. One of the factors whichcould contribute to the reduced inhibitory action of proton-ophores at an alkaline pH is that protonophores become lessefficient as the pH is increased. Protonophores are weakacids, and as the pH is increased above their pK, theproportion of undissociated form to anionic form is reduced.Current theories of the action of protonophores propose thatboth forms of the protonophore are required in the mem-brane to produce a cycle which transports protons across themembrane (22, 23). As the pH is increased above the pK ofthe protonophore, the amount of the undissociated form ableto enter the membrane could become too low to maintainthis cycle (22). TCS, with a higher pK value than CCCP orFCCP (Fig. 3), would have more molecules than the otheruncouplers in the undissociated form at pH 8.5 and hencewould be expected to act more efficiently as a protonophoreat this pH. It has been pointed out elsewhere (19) that CCCPdoes not function well at even a moderately alkaline pH.The possibility always exists that TCS inhibited mem-

brane transport more than the other protonophores becauseof its effects on other metabolic systems. Harold and Baarda(10), however, found that a large number of uncouplers,

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?acoMccc

CF3 FCCP

H

ClTCS

FIG. 3. Structures of the protonophores studied. The pK valuesfor CCCP and FCCP were 6.09 and 5.7 to 6.1, respectively. The pKvalues for two salicylanilides other than TCS were 6.8 to 7.6 (1, 22).

including TCS and CCCP, inhibited energy-dependent mem-brane transport into Streptococcus faecalis. All of the un-couplers accelerated the translocation of protons across thecytoplasmic membrane of this organism and, at concentra-tions of 0.1 to 100 ,uM (0.17 to 170 nmollmg), had little effecton other metabolic systems examined.The moderate halophile and the marine bacteria showed a

greater reduction in sensitivity to protonophores, particu-larly CCCP, than did E. coli as the p-I was increased from7.0 to 8.5. This could have been due to a greater capacity ofthese organisms than of E. coli to produce a AVI at pH 8.5.Tokuda et al. (31-34) proposed that in V. alginolyticus 138-2the At at pH 8.5 is generated by a primary electrogenic Na+pump. Our results, however, show that toluene-treated cellsof V. alginolyticus 118 oxidized NADH at a greatly in-creased rate at pH 8.5 as compared with pH 7.0 in theabsence of added Na+ as well as in its presence (Table 4) andthat the AP generated was greater at pH 8.5 than at pH 7.0(Table 2). The increased AP could have arisen as a result ofan increased extrusion of protons at the various energy-transducing sites in the electron transport chain(s).There was also a wide variation in the sensitivity of the

different organisms to protonophores. E. coli was one of theleast sensitive of the organisms and V. costicola was one ofthe most sensitive at both pHs 7 and 8.5. Such variationscould have been due to differences in membrane composi-tion. Studies with artificial bilayer membranes have shownthat the pH of maximum conductance by an uncouplerdepends on both the uncoupler and the charge on thephospholipid used to prepare the membrane (12).The cell concentrations which have been used to measure

membrane transport, respiration, At, and Na+ extrusionhave varied over a 30-fold range both in our experiments andin those of other investigators for a number of legitimatereasons. If, as has been found with various inhibitors (2, 6,26), a stoichiometric relationship exists between the amountof inhibitor required and the amount of target site present,then a molar concentration of inhibitor which is just suffi-cient to inhibit at a low cell concentration would not inhibitas completely if the cell concentration were increased 30-

fold. From the molarities of inhibitors and our estimates ofcell densities used by the various investigators engaged instudies relating to the Na+ pump, we have calculated theamounts of inhibitors used by each per unit weight of cells inthe various experiments reported. These figures (shown inparentheses after each molarity used) varied from experi-ment to experiment and over a considerable range. If thestoichiometry with cells of other organisms is similar to thatwith V. alginolyticus 118, then a number of the inhibitorconcentrations used by these investigators would not beexpected to be inhibitory.

Results with Na+-loaded cells of V. alginolyticus 118showed that the cells are able to extrude Na+ against agradient at pH 8.5 by a process which is sensitive both torespiratory inhibitors and to protonophores. Thus, it wouldappear that a proton gradient is responsible for the extrusionof Na+ by this organism and that the most likely process bywhich Na+ extrusion takes place is via an Na+/H+ antiportsystem (19). Since TCS prevented Na+ extrusion completelyafter the small initial drop in Na+, there was no evidencethat a TCS-resistant primary electrogenic Na+ pump wasalso operating. Hamaide et al. have observed Na+/H+antiport activity in V. costicola at pH 8.5, and this activitywas prevented by both CCCP and TCS (9).The oxidation of NADH by intact cells of V. alginolyticus

118 and the much higher rate of oxidation of this substrate bytoluene-treated cells (Table 4) are consistent with there beingtwo sites of oxidation of NADH, one on the inside surfaceand one on the outside surface of the cytoplasmic membraneof this organism. In this respect, V. alginolyticus 118 issimilar to A. haloplanktis and other marine bacteria exam-ined previously (18). The requirement for Na+ for' theoxidation of succinate by intact cells but not by toluene-treated cells is indicative of the presence of an Na+-depen-dent transport system for succinate in intact cells such as hasbeen detected in other marine bacteria (5). This transportsystem appears to be appreciably more active at pH 7.0 thanat pH 8.5.

In cells made permeable by toluene treatment, Na+ stim-ulated NADH oxidation, particularly at pH 8.5, but reducedthe rate of oxidation of succinate. This result would suggestthat the site of action of Na+ in the respiratory chain liesbetween NADH and quinone, since from quinone to oxyg,en,electrons from NADH and succinate might be expected toshare the same pathway. Ken-Dror and Avi-Dor (14) andUdagawa et al. (37) have carried out similar experimentswith inverted membrane vesicles of the moderate halophilesBa1 and V. costicola, respectively. They have come tosimilar conclusions regarding the site of action of Na+ in therespiratory chains of these organisms. More definitive ex-periments with V. alginolyticus 138-2 (35) and the moderatehalophile Ba1 (15) have further localized the site of action ofNa+ to within the NADH:(quinone-acceptor) oxidoreduc-tase segment of the respiratory chain. The enzyme in V.alginolyticus 138-2 has a pH optimum of 8 (37). Thus, despiteits sensitivity to protonophores at an alkaline pH, V. algi-nolyticus 118 appears to possess an NADH oxidase which isactivated by Na+ at an alkaline pH. We have observedNa+-stimulated NADH oxidases in other marine bacteriaand have proposed a role for them in exercising metaboliccontrol in the cells (18).

Although our results with V. alginolyticus 118 do notsupport the conclusion of Tokuda and Unemoto (32, 33) thata protonophore-resistant primary electrogenic Na+ pumpoperates in moderate halophiles at an alkaline pH, Tokuda(28) has isolated mutants of V. alginolyticus 138-2 which, at

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pH 8.5, are much more sensitive than the parent cells toCCCP, possess NADH oxidase activity which is not furtherstimulated by Na+, and extrude Na+ by a CCCP-sensitivemechanism. He has concluded that the Na+ dependence ofNADH oxidase represents the Na+ pump. These mutantsrequire further analysis. Are multiple mutations involved,and if so, does one of them lead to increased penetrability ofthe cytoplasmic membrane by CCCP? It is interesting thatthe mutants possess an Na+ extrusion system which, at pH8.5, is sensitive to CCCP. This fact suggests that the Na+extrusion system in the mutants is an Na+/H+ antiporter, asystem not detected in the parent cells, perhaps becauseat pH 8.5 CCCP at the concentration tested failed to estab-lish an efficient proton-shuttling mechanism in the mem-brane.

ACKNOWLEDGMENT

This study was supported by a grant from the Natural Sciencesand Engineering Research Council of Canada.

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