JOURNAL OF BACTERIOLOGY, July 1967, p. 53-60Copyright © 1967 American Society for Microbiology

Vol. 94, No. 1Printed in U.S.A.

Gramicidin, Valinomycin, and Cation Permeability ofStreptococcus faecalisF. M. HAROLD AND J. R. BAARDA

Division of Research, National Jewish Hospital 80206, and Department of Microbiology, University of ColoradoSchool of Medicine, Denver, Colorado 80220

Received for publication 18 March 1967

Gramicidin and valinomycin in concentrations of 10-7 and 10-6 M, respectively,inhibited the growth of Streptococcus faecalis. Inhibition of growth was associatedwith loss of Rb+ and K+ from the cells, and could be reversed by addition ofexcess K+. Cells treated with these antibiotics exhibited greatly increased permea-bility to certain cations; no effect was observed on the penetration of other smallmolecules. Unlike normal cells, cells treated with gramicidin rapidly lost internalRb+ by passive exchange with external cations, including H+, all monovalentalkali metals, NH4+, Mg++, and tris(hydroxymethyl)aminomethane. Exchange wasrapid even at 0 C and was independent of energy metabolism. The effect ofvalinomycin was more selective. Cellular Rb+ was rapidly displaced by externalH+, K+, Rb+, and Cs+; other cations were less effective. The exchange was inde-pendent of metabolism but strongly affected by temperature. Under certain condi-tions, polyvalent cations inhibited exchange between 86Rb and Rb+ induced byvalinomycin. The antibiotic apparently neither stimulates nor inhibits the energy-dependent K+ pump of S. faecalis, but exerts its effect on the passive permeabilityof the membrane to cations. The increased permeability to specific cations inducedby gramicidin and valinomycin is a sufficient explanation for the inhibition ofgrowth, glycolysis, and other processes.

In 1944, Hotchkiss (12) reviewed what wasknown at the time concerning the mechanism ofaction of the related antibiotics tyrocidin andgramicidin (GMCD). He concluded that tyrocidingrossly disrupts the cytoplasmic membrane ofbacteria by a process analogous to th at caused bycationic detergents (17), and this interpretationwas confirmed by more recent work (14). GMCDappeared to act at a different level, inhibitingphosphorylation reactions by a mechanism in-volving K+. Subsequently it became apparentthat GMCD also acts upon membranous struc-tures; it is a powerful uncoupling agent for oxida-tive phosphorylation in bacteria and mito-chondria (6, 18). The structure of GMCD, amixture of closely related linear polypeptides, hasbeen established (21). Very recently, evidence wasobtained that the primary action of GMCD maybe to increase the permeability of the mitochon-drial membrane to cations; the uncoupling ofoxidative phosphorylation appears to be a second-ary consequence of this alteration in membranestructure (3).The cyclic polypeptide antibiotic valinomycin

(22; VAL) also uncouples oxidative phosphoryla-tion under certain conditions (11, 20). The pri-mary effect of VAL, like that of GMCD, appearsto be on cation transport. Addition of VAL torespiring mitochondria stimulates K+ uptake andejection of H+. It is believed that this antibioticincreases the permeability of the mitochondria tocertain cations, and that it may also react directlywith a specific K+ carrier (10, 11 19, 20). Vir-turally nothing is known concerning the effect ofVAL on microorganisms.The present experiments were initiated as part

of a program of research on the transport ofK+ in Streptococcusfaecalis. This microaerophilicorganism derives energy entirely by fermentationof glucose to lactic acid; the absence of oxidativephosphorylation considerably simplifies the inter-pretation of our findings. We shall present evi-dence that the inhibition of the growth of S.faecalis by GMCD and VAL ultimately resultsfrom potassium deficiency. Both antibiotics inter-act with the cytoplasmic membrane to increase itspermeability to cations, and thus interfere withthe selective accumulation of K+.

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MATERIALS AND METHODS

The procedures used in the present study have beendescribed in earlier papers (9, 9a), and a brief sum-mary will suffice here.

Growth media. S. faecalis strain 9790 was usuallygrown on defined media; both K+ and Rb+ supportgrowth (9a, 15). Because of the technical convenienceof 86Rb as an isotope of Rb+, cells grown on Rb+ wereused whenever possible. Glucose served as energysource, and growth was followed by turbidimetry at600 m,u.

(i) Medium NaM is buffered with sodium maleate(200 to 350 mM Na+) and contains various amountsof K+ or Rb+ (9a).

(ii) Medium KM is buffered with potassiummaleate. It contains 200 mM K+ but no Na+.

(iii) Medium TEA is buffered with triethanolamine(15) and is supplemented with K+ or Rb+.

(iv) Medium NaTY is a complex medium (9a, 26)containing 150 mM Na+ and 5 mm K+.

Cation content. Cells were loaded with 86Rb bygrowth or incubation for several hours in media con-taining 86Rb of known specific activity. Under theseconditions, the cells exchange internal Rb+ for 86Rb(9a); at equilibrium the radioactivity of the cells is ameasure of their Rb+ content. Samples were harvestedby filtration on nmembrane filters (Millipore Corp.,Bedford, Mass.), washed with water or 2 mm MgC12,and counted. When desired, K+, Na+, and Rb+ wereextracted and estimated by flame photometry.

Other procedures. Protoplasts were prepared byincubation with lysozyme as described by Abrams(1). Glycolysis was monitored at constant pH byautomatic titration of the lactic acid produced, witha Radiometer pH-stat. In a few experiments, uptakeof H+ by the cells was likewise followed by automaticaddition of standardized HCI at constant pH.

Antibiotics. Gramicidin and tyrocidin were com-mercial products (Nutritional Biochemicals Corp.,Cleveland, Ohio). Valinomycin was a gift from J. C.MacDonald. Antibiotics were dissolved in ethylalcohol; control flasks always received a volume ofethyl alcohol equal to that of the experimental flasksusually 1% by volume.

RESULTS

Inhibition of growth. Cells growing in mediumKM, containing K+ as major cation, were littleaffected by addition of either VAL (5 ,g/ml,5 X 10-6 M) or GMCD (2 Ag/ml, 1 X 10-6 M),though higher concentrations of GMCD did in-hibit growth. However, in media containing pre-dominantly Na+ or triethanolamine, addition ofVAL at 1 ,ug/ml (10-6 M) reduced the growthrate by 80%; GMCD at 0.2 Ag/ml (10-7 M)stopped growth completely. In both cases, addi-tion of excess K+ permitted resumption of growth.Representative experiments of this type are shownin Fig. 1.

Cells exposed to GMCD formed the expectednumber of colonies when plated on KM medium,

A1 A. B.~0.8 3Z 0.6uil 3a0.42-j

0.2-I- * ~~~~2

0.0.1

MCD KCI VALKC

0.51 I I I fj/IAL II.

HOURS HOURSFIG. 1. Effect of gramicidin and valinomycin on

growth of Streptococcus faecalis. (A) An exponientiallygrowing culture in medium TEA was divided (arrow)and the following additions were made: 1) control,ethyl alcohol only; 2) gramicidin (GMCD), 0.2 ,g/ml;3) gramicidin, followed by 0.2 M KC1 at 2.5 hr. (B) Aparallel experiment in medium NaM, with additions asfollows: 1) Control; 2) valinomycin (VAL), I ,ug/ml;3) valinomycin, followed by 0.2 M KCI at 2.5 hr.

but only 0.1% were viable on medium NaM. Itappears that GMCD is not readily dissociatedfrom the cells by dilution. Cells treated with VALformed the same number of colonies on bothmedia. Gross lysis was never observed with eitherantibiotic; this contrasts with the effects of tyroci-din which stopped growth at 1 ,ug/ml (0.8 x10-6 M) in all the media tested and also inducedvisible lysis.

Experiments with S. faecalis growing inmedium NaM-86Rb provided direct evidence foran effect of VAL and GMCD on the metabolismof inorganic cations. During the exponential phaseof growth, untreated cells contained some 700,umoles of 86Rb per g (dry weight) of cells. Within10 min, the 86Rb content fell to 300 ,umoles/g uponaddition of VAL (1 ,g/ml) and to 30 ,moles/gwith GMCD (0.2 ug/ml). These results suggestthe interpretation to be supported below, thatboth antibiotics interfere with the selective ac-cumulation of K+ and Rb+; growth of the cellsthen becomes dependent upon high extracellularconcentrations of these cations.

Retention of K+ and Rb+ During exponentialgrowth, S. faecalis contains up to 800 ,moles ofK+ or Rb+ per g of cells, together with smallamounts (50 to 150 umoles/g of cells) of Na+. Ina series of measurements, the ratio of total mono-valent cations to total phosphorus was 1.0 to 1.3,suggesting that a large fraction of the cations maybe associated with cellularphosphorus compounds.

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VOL. 94, 1967 GRAMICIDIN, VALINOMYCIN, AND CATION PERMEABILITY

Cells loaded with K+ or 86Rb may be washedrepeatedly with water, KC1, or NaCi (0.1 M)without appreciable loss of 86Rb. However, cellstreated with GMCD or VAL lost 86Rb to varyingdegrees upon washing with dilute solutions of salts(Table 1). In cells treated with GMCD, 86Rb waslost upon washing with all the cations tested.The effect of VAL was more selective. 86Rb wasdisplaced much more rapidly by K+, Rb+, andCs+ than by the other cations. The displacementof 86Rb is due to exchange with cations from thewash fluid, as will be shown below. Entirelyanalogous results were obtained with protoplasts,pointing to the cytoplasmic membrane as thetarget organelle of these antibiotics.

It was shown above that high concentrations ofK+ nullify the inhibition of growth by both VALand GMCD (Fig. 1). Two lines of evidence sup-port the conclusion that 0.2 M K+ does not inter-fere with the action of the antibiotics per se, butmerely makes good the loss of K+ from the cells.Cells were loaded with "6Rb and suspended in0.2 M KCI. Upon addition of the antibiotics(1 jg/ml), the "Rb was instantaneously dis-placed. S. faecalis was grown in medium TEAsupplemented with 0.1 M 88Rb. Addition of VALor GMCD had little effect on growth. However,

TABLE 1. Release of 86Rb by Streptococcus faecalistreated with valinomycin or gramicidina

Percentage of '6Rb retained

WashEthyl Vali- Gramicidinalcohol only nomycin Gaiii

Water.100 87 59LiCl, 20 mM . 100 73 0.5NaCl, 20 mi . 94 62 1KCl, 20mM.95 9 0.5RbCl, 20mM.97 11 0.5CsCl, 20mM.. 90 13 1.5NH4Cl, 20mM.95 61 0.5MgCl2, 20mM.98 67 4.5Tris chloride, 20 mm.. 95 77 22

aS. faecalis was loaded with 86Rb as describedin the text. The cells were washed and resuspendedin water at 0.5 mg/ml. To portions of this sus-pension were added valinomycin, gramicidin(1 jug/ml final concentration), or ethyl alcoholalone (1% final). After 5 min at room temperature,1-ml samples were filtered and washed on thefilter three times, for 1 min at a time, with 2 mlof the various salt solutions. The original cellswashed with water contained 6,100 counts permin per mg of dry cells. This is taken as 100%,and retention of Rb 86Rb is expressed as the per-centage of 86Rb remaining in the cells after ex-posure to ethyl alcohol or antibiotics and wash-ing.

cells harvested from such cultures lost "Rb uponwashing with salts (see Tahle 1), whereas normalcells retained 86RhCation retention by S. faecalis was exquisitely

sensitive to GMCD and VAL. Exposure of cellsunder the conditions shown in Table 1 to as littleas 0.05 ,ug of GMCD per ml (2 X 10-8 M) or0.2 Mug of VAL per ml (2 X 10-7 M) resulted insignificant loss of "Rb. By contrast, the followingagents were without effect: ethyl alcohol, 0.25 M;n-butanol, 0.15 M; polymyxin B, 25 ,g/ml; strep-tomycin, 103 M; Triton x-100, 10 Ag/ml; cetyltri-methylammonium bromide, 2 x 10-i M; dodecyl-trimethylammonium bromide, 3 X 10-5 M;sodium lauryl sulfate, 3 X 10-5 M; n-dodecyla-mine, 5 X 10-5 M. The four ionic detergentscaused lysis of growing cells at concentrationsabove 1 X 10-4 M.

Other effects on membrane permeability. Toassess the effects of VAL and GMCD on theretention of other compounds, cells were loadedwith 32P-orthophosphate (Pi), 14C-alanine, or4C-glutamate by incubation in the presence ofglucose. Chloramphenicol (50 Ag/ml) was addedto prevent incorporation of "4C-amino acids intoproteins. The cells were washed, suspended inwater, and exposed at room temperature to VALor GMCD (1 Ag/ml). The labeled compoundswere not displaced upon subsequent washing ofthe cells with water, buffers, or with solutions ofthe unlabeled compounds. This finding agreeswith the much earlier conclusion of Gale andTaylor (7) thatGMCD does not cause generalizedmembrane disruption.

Additional evidence against gross effects onmembrane permeability was obtained by measur-ing the effect of VAL and GMCD on the stabilityof protoplasts (1, 2). A suspension of protoplastswas diluted into solutions of arabinose, galactose,sucrose, raffinose, glycine, alanine, and valineranging in concentrations from 0.1 to 0.5 M.Turbidities were measured after 2 hr. The finalreadings obtained were not materially altered bythe addition of either antibiotic. By contrast,tyrocidin at 1 Mg/ml caused lysis of protoplastsstabilized by sucrose. By these criteria, then VALand GMCD selectively increase the permeabilityof the membrane to cations, but not to othersmall molecules.

Effect on glycolysis andpermeability to H+. Theeffect of the antibiotics on glycolysis by S. faecalisin water proved to be a function of pH (Fig. 2).At pH 7.5, addition of VAL had no effect. At pH6, VAL stopped glycolysis, but the inhibition wasreversed by addition of KCI or RbCt (1 mM) andof higher concentrations (10 to 100 mM) of NaCIor MgCl2. The effects of GMCD were similar butmore drastic: at pH 7.5, addition of GMCD

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GRAMICIDINkXI M

- /pH 7.5- GMCD

/ /pH 6

K,IOmMMCD t K.IOOmM4 t .4.,

0 4 8 12 14

0 MINUTES

FIG. 2. Effect of valinomycin and gramicidin on

glycolysis. Streptococcus faecalis was harvested duringgrowth on medium KTY. The cells were washed andsuspended in water at I mg/ml. Glucose (10 ,umoles)was added and glycolysis followed on the pH-stat atpH7.5 or 6. Antibiotics were added to I Mg/ml at arrows,

followed by KCI tofinal concentrations shown. Abbrevi-ations: valinomycin, VAL; gramicidin, GMCD; KCI,K.

slowed down the rate of glycolysis, which was

restored by cations at 1 mm. At pH 6, glycolysiswas completely blocked, and high concentrationsof KCI or NaCl were required to elicit its resump-tion.A clue to an understanding of these phenomena

was provided by the observation that addition ofVAL or HMCD to a suspension of S. faecalis inwater was followed by an increase in the pH;considerable amounts of acid were required tomaintain thepH at 6. In the experiment illustratedin Fig. 3, the antibiotics were added to a suspen-sion of cells loaded with 86Rb of known specificactivity, and a pH of 6 was maintained on thepH-stat by addition of standardized HCI. At theend of the experiment, the cells were filtered andthe filtrates were analyzed for 86Rb. The amountof 86Rb displaced from the cells war stoichio-metrically equal to the amount of H+ consumed,pointing to an uptake of H+ in exchange for Rb+.

It would thus appear that VAL and GMCDincrease the permeability of the membrane to H+,which is largely excluded by normal cells (25).H+ enters the cells in exchange for Rb+, inhibitingglycolysis by lowering the internal pH (25). Highconcentrations ofK+ or Na+ displace H+ by massaction, thus restoring glycolyls. The expected dis-placement of H+ by K+ and Na+ from cells ex-

posed to VAL or GMCD under the conditions ofFig. 3 was readily observable by use of the pH-stat. Displacement of over 90% of the 86Rb byNa+ did not reduce the rate of glycolysis.

Cation exchanges induced by VAL and GMCD.The cation exchanges induced by VAL andGMCD were examined with respect to theirspecificity and temperature dependence. Cellswere loaded with 86Rb, washed, and resuspendedin 10 mm NaCL or RbCI at various temperatures;the pH was adjusted to 7.5 (to minimize avail-ability of H+), VAL was added (1 Ag/ml), andsamples were taken at intervals. Results are shownin Fig. 4. Displacement of 86Rb by Rb+ was com-pleted in 2 min at 37 and 20 C and much moreslowly at 0 C. Na+ displaced at 37 and 20 C,although more slowly at 20 C, and had littleeffect at 0 C. The exchange was approximatelystoichiometric. In a particular experiment at37 C, 400 ,umoles of K+ per g of cells were lost,whereas 250 ,umoles of Na+ were gained (thediscrepancy may reflect the exchange betweenK+ and H+ as discussed above). Results similarto those of Fig. 4 were obtained with other cat-ions: K+ and Cs+ behaved like Rb+, whereasLi+, Mg++, and the cationof tris(hydroxymethyl)-aminomethane (Tris) resembled Na+.Exchange of 86Rb by cells treated with VAL

at 0 C was inhibited by other cations, particu-larly polyvalent ones (Fig. 5). Cells were loadedwith unlabeled Rb+, washed, and resuspended inwater at 0 C, atpH 7.5. VAL was added (1 jug/ml), followed by 86Rb at 0.1 mm. Under theseconditions, 86Rb entered the cells by exchange forunlabeled Rb+; the total Rb+ content of the cellsremained constant. The exchange was inhibited

~2.0zx i.5-

i I.0 22-Jo 0.5 0

0 2 4 6 8

MINUTESFIG. 3. Induction of exchange between H+ and 86Rb

by valinomycin and gramicidin. Cells loaded with 86Rbof known specific activity were suspended in water at0.7 mg/ml. Samples of the suspension were adjusted topH 6, and additions were made at 0 min: 1) gramicidin,I ,Ag/ml; 2) valinomycin, I ,ug/ml; 3) ethyl alcohol only.The pH was maintained at 6.0 on the pH-stat by use of0.011 M HCI; the lines represent H+ consumption. Atthe end, the cells were filtered and the filtrates analyzedfor 86Rb. The amount of86Rb lost from the cells is shownby the circles.

56

(n4J

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\ 0.4

Cl)Lii 0.30;; 0.2

0*

J. BACTERIOL.

VALINOMYCIN

-VAL

I/pH 6

pH 7.5

- KKA mM lOmM

4 4.4I,0 4 8 12

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VOL. 94,1967 GRAMICIDIN, VALINOMYCIN, AND CATION PERMEABILITY

1000

LAJ

U 500

0 0 1 00 5 1 5 2

200

d iCRb i, 20mM0 37*

50 *20"

I-ml samp eNoClt,0imM el

200

.a..0

0 5 15 ~20 0 5 10 15 20

MINUTES MINUTES

FIG. 4. Effect of temperature upon the displacement

Of86Rb froM cells treated with valinomycin by NaCI and

RbCl. CellS loaded With 86Rb were washed and resus-

pended in 10 mms NaCI or RbCl at 37, 20, and 0 C.

Vailnomycin (1 jig/ml) was added at 0min At intervals,1-ml samples were filtered, and the 86Rb in the cells was

determined. The initial 86Rb content was normalized to1,000 counts per min per mg of cells.

by NaCI (5 mM) and virtually abolished by Mg++(1 mM), spermine, spermidine, and streptomycin(0.2 mM). Inhibition of exchange between 86Rband Rb+ by polyvalent cations was much lesspronounced at higher temperatures.

Similar experiments were carried out withGMCD. In this case, displacement of 86Rb by allthe cations listed in Table 1 was essentially com-plete in a few minutes, even at 0 C. The exchangeswere again approximately stoichiometric and werebut little affected by polyvalent cations (whichthemselves displaced 86Rb).

Inhibition of energy-dependent K+ transport.The results presented thus far indicate that VALand GMCD increase the permeability of the mem-brane to cations. To determine whether the anti-biotics react specifically with the transport systemfor Rb+ and K+ (9a), we investigated the effectof the antibiotics upon energy-dependent net up-take of K+.CeUs grown overnight on medium NaTY are

relatively depleted of K+, but contain largeamounts of Na+ and H+. In the presence of glu-cose, such cells take up K+ with concurrent extru-sion of Na+ and H+ (9a, 26). VAL (1 ,ug/ml) hadlittle effect upon the uptake of K+ from water atpH 7.5. (Fig. 6). In some experiments, the K+ thatinitially accumulated was later displaced again, asNa+ was added to neutralize the lactic acidformed during glycolysis.A number of controls were required to buttress

the conclusion that VAL does not affect theenergy-dependent net uptake of K+. The net

uptake of K+ and the extrusion of Na+ weredependent upon glucose both in the absence ofVAL and in its presence. Moreover, extrusion ofNa+ (and, of course, uptake of I6+) occurred onlywhen K+ was provided, both in the absence ofVAL and in its presence. It was shown in previous

0LLI

z

X

Li

0D0ID

8

60

40

20

10

0 5 10 20

MINUTESFIG. 5. Induction ofexchange between 86Rb and Rb+

by valinomycin at 0 C, and its inhibition by othercations. Cells were loaded with Rb+, washed, and resus-pended in water at 0 C. Valinomycin was added (I,g/mg). After 5 min, additions were made as shown,followed by 88Rb (0.1 mM). Samples were filtered atintervals, washed, and counted. Control suspensions notexposed to valinomycin (omittedfor clarity) took up no

measurable 86Rb.

'I)

1000vs

0'

\ 800

6000

o

0 5 10 200 5 10

MINUTES20

MINUTES

FIG. 6. Effect of valinomycin on energy-dependentnet uptake of K+. Cells were grown on NaTY, washed,and resuspended in water. Glucose was added andglycolysis initiated at 37 C, pH 7.5. Ethyl alcohol orvalinomycin (I pAg/mI) was added, followed 3 min laterby 2 mm KCI. Samples were filtered, washed, andanalyzedfor K+ and Na+.

CONTROL VALINOMYCIN

o No0

0-0 0 . .-9

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HAROLD AND BAARDA

sections that cells treated with VAL at pH 7.5 be-came permeable to K+ and Rb+, whereas Na+entered more slowly. Thus the success of thepresent experiment depends upon careful choiceof conditions. The pH must be high to minimizeavailability of H+, and the Na+ content of themedium must be kept low to minimize displace-ment of K+. At pH 6, VAL completely blockednet uptake ofK+ from water.As expected, parallel experiments with GMCD

gave equivocal results. Even in the absence ofglucose, 1 mm KCI passively displaced Na+ (andvice versa). It was therefore impossible to deter-mine whether or not the energy-dependent K+transport system continued to function in thepresence of GMCD.

Inhibition of energy-dependent transport of 32p_Pi andamino acids. The effect ofVAL andGMCDupon the transport of 32P-Pi and amino acids wasfound to depend upon the cation composition ofthe medium. Cells of S. faecalis were suspendedin either sodium maleate or potassium maleate atpH 7, 37 C (chloramphenicol, 50 gg/ml, wasadded to suspensions which later received'C-amino acids). The suspensions were pre-incubated with glucose for a few minutes; VALor GMCD were then added, followed by radio-active substrate. At these concentrations, bothNa+ and K+ permitted glycolysis to continue.

Results for 32P-Pi and 14C-glutamate are shownin Table 2. Neither VAL nor GMCD inhibiteduptake by cells in potassium xmaleate, but bothwere inhibitory in sodium mateate. It is probablethat inhibition of uptake by the antibiotics is in-direct and mediated via their effects on cationpermeability.

DiscussioN

S. faecalis accumulates large-amounts of K+and Rb+ during growth (9a), apparently in asso-ciation with various phosphorylated compounds.These cations are tenaciously retained and ex-

change but slowly with external cations in theabsence of a source of metabolic energy (9a).The permeability barrier to cations presumablyresides in the cytoplasmic membrane, since cat-ions are lost when the cells are treated withagents known to disrupt the membrane such as

organic solvents, detergents, or tyrocidin (4, 7,8). The cation barrier is exceedingly sensitive tothe antibiotics GMCD and VAL. These sub-stances in concentrations of the order of 108 to10-7 M markedly increase the rate of exchange ofcations across the membrane, but (unlike the non-specific agents enumerated above) have no ap-parent effect on the permeability to other small

TABLE 2. Inhibition of the uptake of 32P-Pi and4C-glutamate by valinomycin and gramicidina

Medium Additions Cglu 2ppictamate6

Potassiummaleate..... ... Ethyl alcohol 1,765 3,890

onlyPotassiummaleate........ Valinomycin 1,685 3,495

Potassiummaleate........ Gramicidin 1,785 3,300

Sodium maleate.. Ethyl alcohol 1,900 2,450only

Sodium maleate.. Valinomycin 970 1,000Sodium maleate.. Gramicidin 150 1,370

a Streptococcus faecalis was grown on mediumKM. The cells were washed and suspended in0.1 M sodium maleate or potassium maleate atpH 7, 37 C. Glucose was added and the cells wereincubated for 5 min. To samples of the suspensionwere added valinomycin (1 pg/ml), gramicidin(1 pg/ml), or ethyl alcohol alone; 3 min later,32PjPi (3 X 10-4 M) or 14C-glutamate (1.3 X 10-M) were added. The latter suspension also con-tained 50 jig of chloramphenicol per ml.

bExpressed in counts per minute per milligramof cells after 2 min.

- Expressed in counts per minute per milligramof cells after 10 min.

molecules. It must be stressed here that cationexchanges induced by GMCD and VAL do notrequire an exogenous substrate and take placeeven at 0 C. S. faecalis apparently lacks internalenergy reserves, since transport of 32pi, 1C-glutamate, 86Rb, and K+ by normal cells is strictlydependent upon an exogenous substrate (9, 9a,26); thus, the cation exchanges induced by theantibiotics must be metabolically passive.Of the two antibiotics, GMCD has the more

drastic effects. Even at 0 C, GMCD-treated cellsrapidly exchange internal Rb+ or K+ for externalcations ranging from H+ to Mg++, and moreslowly for bulky cations like Tris and spermine.These results agree well with the findings ofChappel and Crofts (3) in mitochondria.The effects of VAL are more subtle. In the

presence of VAL, 86Rb exchanges rapidly for H+,K+t Rb+, and Cs+, more slowly for Na+, Li+,Mg++, and the cation of Tris. This suggests aninverse relationship between the rate of entry ofthe cation and its hydrated ionic radius (23).The rate of exchange also depends strongly uponthe temperature. At 0 C, cation exchanges arestrongly inhibited by polyvalent cations which arebelieved (8, 24) to react with the membrane. Thekinetics of exchange are complex and do not fit a

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single exponential (Fig. 4, 5); detailed kineticanalysis was not carried out.

Previous studies (9a, 26) on cation uptake byS. faecalis have furnished evidence for the exist-ence of specific transport systems which mediateselective, energy-dependent uptake of K+ andRb+. From the results presented here (Fig. 6),it appears that VAL neither stimulates nor in-hibits this K+ pump, but rather affects cationpermeability of the membrane in general. Thisfinding differs from that of Pressman and co-

workers (10, 11, 20) who concluded that VALstimulates the K+ pump of mitochondria. Wewere unable to determine whether the K+ pumpcontinues to function in the presence of GMCD.The increased permeability of the membrane to

cations is a sufficient explanation for the inhibi-tion of growth by VAL and GMCD and itsreversal by K+. The action of GMCD and VALis thus reminiscent of that of certain polyeneantibiotics on fungal cells (4). A high internalconcentration of K+ is required for growth ofS. faecalis (9a) and of Escherichia coli (13). In E.coli, the process most sensitive to K+ deprivationis protein synthesis (13). The situation S. faecalismay be more complex; although glycolysis doesnot require K+ (1, 2), a variety of transport proc-esses appears to be impaired in cells deficient ofK+. The present findings confirm the involvementof K+ in the uptake of amino acids (5, 16), sugars(2), and phosphate (9), and provide a tool whichmay help clarify the role of K+ in these diverseprocesses.

ACKNOWLEDGMENTS

We thank J. C. MacDonald, Prairie RegionalLaboratory, Saskatoon, Saskatchewan, Canada, forhis generous gift of valinomycin, and A. Abrams forcritical discussions and the loan of his pH-stat.

This investigation was supported by Public HealthService Grant AI-03568 from the National Instituteof Allergy and Infectious Diseases.

LITERATURE CITED

1. ABRAMS, A. 1959. Reversible metabolic swellingof bacterial protoplasts. J. Biol. Chem. 234:383-388.

2. ABRAMS, A. 1960. Metabolically dependent pene-tration of oligosaccharides into bacterial cellsand protoplasts. J. Biol. Chem. 235:1281-1285.

3. CHAPPELL, J. B., AND A. R. CROFTS. 1965. Gram-icidin and ion transport in isolated mitochon-dria. Biochem. J. 95:393-402.

4. CIRILLO, V. P., M. HARSH, AND J. 0. LAMPEN.1964. Action of the polyene antibiotics filipin,nystatin and n-acetylcandidin on the yeast cellmembrane. J. Gen. Microbiol. 35:249-259.

5. GALE, E. F. 1953. Assimilation of amino acids bybacteria and the action of some antibioticsthereon. Advan. Protein Chem. 8:285-391.

6. GALE, E. F. 1963. Mechanisms of antibioticaction. Pharmacol. Rev. 15:481-530.

7. GALE, E. F., AND E. S. TAYLOR. 1947. The assimila-tion of amino acids by bacteria. II. The actionof tyrocidin and some detergent substances inreleasing amino acids from the internal environ-ment of Streptococcus faecalis. J. Gen. Micro-biol. 1:77-84.

8. HAROLD, F. M. 1964. Stabilization of Strepto-coccus faecalis protoplasts by spermine. J.Bacteriol. 88:1416-1420.

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