Binding of lipophilic anions to microbial cells

ELSEVIER

Bio¢lectrochcmistryand Bioenergetics42 (1997)263-274

Binding of lipophilic anions to microbial cells 1 Rimantas DaugelaviEius a.c, Elena Bakien6 a, Janina Ber~inskien6 a, Dennis H. Bamford b.c., Department of Biochemistry and Biophysics, Viinius University, Ciurlionio 21, LT-2009 Vilnigs. l.,ith+~ania u Institute of Biotechnology, P.O. Box 56 ~V~ikinkaari5). Unicersity of Helsinki, FIN.O0014Hebinki, Finland c Department of Biosciences, Division of Genetics, p.o. Box 56 fViikinkaari 5). Unieersiff of Helsinki. FIN-O0014Helsinki, Finland

Absti~ We have studiedthe binding of lipophilicanions (LPAs) to bacterialand yeast cells,E. co/i-derivedmembrane vesiclesand pete phospholipidvesiclesand membnme-contalning bacteriophageparticles,The inactivationof the cellsby heator phenol treaem,m leadsto a sixfoldto eightfoldincreasein the binding of phenyldicarbanndecaborane(PCB-). However, heatinghas a small effecton the suoag binding of this anion to membrane vesicles and membrane-containing phage particles. Similar to l~B-, the imact cells b~mt sm~ amounts of naphthyldicarbaundecaborane (NCB-) and tewapbenylboron(TPB-). However, the cells bind 6--15.fold larger ~ of diphenyldicad~anndecaborane (DIN2B-) and decyldicarbaundecabor,~ (DCB-) than PCB-. The binding of DCB- a ~ ~ to membrane-containing phage particles, pure lipid vesicles mid a stabilized emulsion of olive oil is also s~'onger relative to the b i ~ of I~B-. The results obtained with the polycationic antibiotic Polymyxin B (PMB) and bacterial viruses suggest that the smaller an',ot~ of binding of LPAs to intact cells is at least partially due to the difficult partitioning into the interinr of phospholipid bilayers. PMB a tenfold increase in PCB- binding to intact E. coil cells. It also increases PCB- binding to beabinactivated cells (about 15%) and to envelope-containing viruses (up to 60%), This indicates the presence of an intrinsic barrier for LPA binding to membranes. Screen/rig of the cell surface charge by high concentrations of salts affects the binding of LPAs rather weald1',but the amount of buund LPAs irg'reases considerably when the medium is below pH 4. © 1997 Elsevier Science S.A. Keywords: Lipophiticanions; Membranebinding;Outer membranepermeability;Phenyklicarbanndeca~mrane;Po]ymyxinB

1. Introduction

Lipophilic ions are a class of charged molecules with the unique property that they are soluble in both hydrophilic and hydrophobic solvents. The best-known examples of lipophilic ions are the indicators of membrane voltage (transmembrane difference of the electrical potential Aqp), e.g. teuaphenylphosphonium (TPP+), tetraphenylboron (TPB-), phenyldicarbaundecaborane (PCB-) and their analogues. Over 25 years ago, Liberman et al. [1] showed that energized mitochondria accumulated lipophilic cations, but not anions, whereas submitochondrial vesicles accumulated lipophilic anions (LPAs), but not cations. These results were compatible with the chemiosmotic hypothesis, according to which mitochondria and bacteria generated a negative membrane voltage, ' Correspondingauthor. Tel.: 358-0-70859100;fax: 358-0-70859098; e-mail: [email protected],fi. ' Presentedat the 13th International Symposiumon Bioclectrecheraistry and Bioenergetics,Ein Gedi, Israel, 7-12 January 1996. 0302-4598/97/$17.00 © 1997ElsevierScienceS.A. All fights ~servecl. PI! S0302-4598(96)05096-9

whereas submkochondrial and bacterial vesicles with an

inverted membrane polarity generated a positive membrane voltage. Measurement of the membrane voltage hi isolated mitochondria and microbial cells by lipophHic cation distribution has become one of the central techniques in bioenergetics (for reviews, see Refs. [2,3]). In~eractions of lipophilic cations wRh microbial and mitocho~

drial membranes have been s~ied extensively [4-7]. LPAs have mostly been used to study the energetics of submitocbondrial and bacterial membrane vesicles [8,9], to increase the permeability of cellular memO:fanes to lipophilic cations [5,10] and to investigate the winciples of membrane permeability using liposomes [l 1,12] and planar lipid membranes [13,|4]. Biological membranes are permeable to lipophilic ions due to their ability to bind and easily translocate across lipid bilayers. A large ionic size and a delocalized electric charge, as well as the presence of hydrophobi: groups. appear to be the necessary attributes for enhanced permeability. All of these factors are expected to decrease the energy required to transfer the ion from the aqueous phase

264

R. Daugelavi~iuset al./ BioelectrochemistryandBioenergetics42 (i9~7) 263-274

to the non-polar membrane phase having a low dielectric constant. In early studies of the transport of lipophilic ions across lipid bilayers, it was observed that LPAs interact with membranes in a different manner from structurally similar cations [15], even when very close analogues such as TPP + and TPB- are used. It was observed that LPAs bind to membranes several orders of magnitude more strongly and translocate across bilayers several orders of magnitude more rapidly than structurally similar cations [16,17]. Differences in the adsorption of positively and negatively charged lipophilic ions have been successfully explained by the dipolar model of lipid bilayer membranes developed by Flewdling and Hubbell [18]. Binding regions for the !ipophilic ions are present near membranelwater interfaces, where the potential energy within the membrane is at a minimum. The anions exhibit a deeper binding minimum resulting in much stronger binding compared with. the cations. It is obvious that the dipole potential, produced by molecular dipoles at membrane[solution interfaces [19], is a main factor in determining the high LPA binding capacity of phospholipid monolayers and bilayers [12,18]. In most cases, the interaction of LPAs with intact microbial cells has been investigated indirectly using anionic fluorescent probes [20,21] or uncouplers of oxidative phosphorylation (for a review, see Ref. [22]). The deprotohated forms of the uncouplers can be considered as LPAs. In attempts to investigate the interaction of TPB- with Tris/EDTA-treated E. coli cells [23], it remained unclear why the cellular membranes bound only low amounts of this LPA. We demonstrate here that a cell envelope permeability barrier for LPAs exists in live microbial cells.

2. Experimental details 2.1. Chemicals

2.2. Biolegical material and growth conditions B, subtilis T2 (uvr, trp3) cells as well as E. cvli ANiS0 (F-, urgE3, thi, mtl, xyl, str704), with wild-type cell envelope, and E. coli KO1489, a sodium dodecyi sulphate-sensitive derivative of MC4100 (aruD139, A( argF-lac )U169, rpsL150, reiA 7, deoC l , ptsF25, rbsR, thi, supF, Zla:tnlO sdslr), cells were grown in LB medium [24]. Overnight cultures were diluted with fresh medium and grown at 37 °C with aeration from 1 × 10a cells mi-r to 1 X 10 9 cells ml -t before harvesting. Saccharomyces cerevisiae P63-DC5 ( ade2, leu3, his3) cells were grown at 30 °C with aeration in a medium composed of 2% glucose, 2% peptone and 1% yeast hydrolysate from 1 × 10 4 tO 3 x 10 7 cells ml -j . Tris/EDTA-treated E. coli cells were prepared by incubating the bacteria in 100 mM Tris containing 10 mM EDTA, pH 8.0, at 37 °C for 10 min, as described in Ref. [25], Bacteriophage T4 was grown and the number of infectious particles was determined as described previously [25,26]. T4 was concentrated from cell !ysates and purified using a two-phase system (polyethylene glycol 6000/dextran sulphate) as described in Ref. [27]. Bacteriophages PRDI and d~6 were grown, concentrated and purified as described previously [28]. 2.3. F'reparation of membrane vesicles The membrane vesicles of E. cull AN180 cells were prepared by ultrasonic oscillation of the bacterial spheroplasts, as described in Ref. [29]. Protein concentrations were determined according to Bradford [30]. Pure phospholipid vesicles were prepared by injecting 10-50 Isl of the phospholipid solution in ethanol into an appropriate assay buffer containing LPAs. A suspension of very small liposomes (about 25 nm in diameter) was obtained using this method [31]. 2.4. Calculations of partition coefficients

Tetraphenylphosphonium (TPP +) chloride and sodium tetraphenylboron (TPB-) were purchased from Chemapol and Fluka respectively. Potassium phenyl-7,8-dicarbaundecaborane (PCB-), potassium dipbenyldicarbaundecaborane (DPCB-), potassium diiodophenyidicarbauudecaborane (DIPCB-), tetramethylammonium dibenzyldicarbaundecaborane (DBCB-), potassium a-naphthyldicarbaundecaborane (NCB-) and potassium 7-(l-decyl)-dicarbaundecaborane (DCB-) were generous gifts of Dr. L.I. Zakharkin (A.N. Nesmeyanov Organoelement Compounds Institute, Moscow). Polymyxin B sulphate (PMB, 7730 units of Potymyxin B base per milligram) and Gramicidin D (GD) were purchased from Sigma. Phosphatidylethanolamine (PE, P8068), pbosphatidylglycerol (PG, P8318), cardiolipin (CL, C1649) and l-oetanol were also purchased from Sigma. Olive oil and stabilized olive oil emulsion were purchased from Sigma Diagnostics.

Partition coefficients were calculated according to the formula Q = (~bo..Jvo/cf,,:o

where ~ , , , d is the amount of LPAs bound, Vt is the volume of lipids or hydrophobic solvent and Cfre¢ is the concentration of LPAs left in the medium. The total membrane volume of a typical E. coil cell is 0.083 iLm3 assuming a width of 7.5 nm for the two membra~es [32]. This corresponds to 8.3 × 10 -2 ~! of membrane in 109 cells. Using an intermediate value of 1 : 1 for the protein:lipid ratio in both membranes, the lipid phase volume of 4.15 × 10 -2 ILl per 10 9 cells is obtained. The diameter of the membrane vesicle of bacteriophage PRDI is about 50 nm [33] and every phage particle contains about 12000 molecules (1.5 × 10 -~7 g) of lipids

R. Daugelaei[iuset al,/ Bioelectrochemisto'and Bioenergetics42 (1997)263-274 [34]. This corresponds to about 15 ~g of lipids in 10 ¢2 virus particles. The diameter of the phage 66 membrane vesicle is 82 am [35] and it contains about 25000 lipid molecules [36,37]. This corresponds to 3.1 × 10 -17 g of iipids per particle, or 31 ~g of lipids in 10 ~'- virus particles. For the determination of the lipid/water partition coefficients, the volume of the lipid phase was calculated on the basis of a density of 1.0 g mi- ~.

3. Results 3.1. PCB- binding is greatly increased by cell inactivation We examined the uptake of PCB- by both Gram-positive and Gram-negative bacteria and yeast cells. Intact S. cerevisiae, B. subtilis and E. coli cells show only a weak uptake of PCB- (Fig. I(A)Fig. I(B)Fig. i (C), curve I). When these cells are inactivated by incubation at 75°C or by using 7% phenol for 5 rain, PCB- binding is greatly increased (Fig. I(A) Fig. I(B) Fig. I(C), curves 2 and 3, 3 and 4 and 5 respectively). Qualitatively similar results are obtained using intact and heat-killed Citrobacter, Klebsiella, Salmonella, Pseudomonas and Streptococcus cells (not shown). The Outer Membrane (OM) of Gram-negative bacteria is only marginally permeable to lipophilic compounds and the use of iipophilic cations and ionophoric antibiotics for A~ studies requires the permeabilization of the cells by Tris/EDTA treatment [23,25,39]. The experiments with Tris/EDTA-treated E. coil cells show that they bind more PCB- than cells with intact OM, but only about 30% of the amount compared with heat-inactivatod cells (Fig,

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2.5. Assays for ion fluxes For the LPA uptake experiments, the cells were pelleted by centrifugation, washed and resuspended in 100 mM sodium phosphate buffer, pH 7.0. About ( 2 - 3 ) × 10 jt viable E. coli or B. subtilis cells or i × 10 ~° S. cerevisiae cells were resuspended in 1 ml of this buffer and kept on ice until used (within 4 h). An aliquot of 5-80 gl of this mixture was added to an appropriate buffer in a thermostatically controlled reaction vessel (volume, 5 ml) and the cell suspension was aerated by magnetic stirring. The concentrations of LPAs in the medium were monitored using lipophilic ion-selective electrodes, connected to Orion 520 A pH/ISE meters. The electrodes were coustructed using poly(vinyl chloride) (PVC)-based sensors and TPPTPB as an active complex as described in Pet's. [23,38]. These electrodes exhibited a nernstian response, 58 mV per decade at 25 *C, from 2 × 10 -7 to 10 -4 M of PCB-. Ag/AgCI reference electrodes (Orion Research Inc., model 9001) were indirectly connected to the measuring vessels through agar-salt bridges.

265

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Fig. I. Uptakeof PL-"B- by intact~d inactivatedmictotfialcells lind E call membranevesicles.The experimentswere carriedo~ at 2~ ~C (A a,,d D) or 37 *(2(B and C), The cells were i n c ~ in 100 t~M sod/ran phosphate, pH 7.0, and the vesicles in 10 mM Tris/HC1, pH 7.0, conlalning 10 raM MgCIz. S. cerevisiae cells were added to a fi~l concentrationof 2.8× 107 ml- t 8. s~tilis cellsto 9× 10s m]- i E. co/i ANIS0 cells to 6× 10~ ral-~ and E. co/i merabra~ vcfa:Ics to 49 ~.g protein ml- '. A-C curve I. intactcells: A ctnwe 2, B curve 3 aml C curve 4. heat-inactivatedcells; A cadre 3 and C cuc'~e5, !R~.a~-trcag~ cells; B curve2, in the presenceof lysozyme(finalcoexen~rafio~IGOI~g ml~' ); C curve2, TrLs/EDTA-treatedcells;C era-re3, in lhc ~ of phage T4 (final concenWalion.1.8× 109 infectivepaak:les ml-l~ D curve 1. intactvesicles:D ct~e 2. heat-treatedvesicles.

I(C), curve 2). However, membrane vesicles ol~ined from E. coZi cells by ultrasonic disruption ~ s,,~roplasts bind large amounts of PCB- before arid after heating (Fig. I(D)). The lysozyme-inducod lysis of B. sabtilis cells also induces extensive PCIi- binding to the cellular d e n s (Fig. l(B), curve 2), Strong binding of PCB- to E. coli cells is also induced by phage 'I'4 infection (Fig. I(C), curve 3). PMB alone or in a mixture with GD effectively increases PCB- binding to E. coli cells (Table I). PMB, as well as the infection of the cells by phage T4, induces a depolarization of the plasma membrane (PM) [25,26,40,41]. However, the decrease in Aqt alone is not sufficient to ino~ease PCB-

Table I Bindingof lipophilicanionsby E. coli cell~ Adon PCBNCBDPCBDBCBDCBD1PCBTPB-

Amountbouad/nmo!per 1.5× 109cells Intactcells

After~klitioaof PMBand GD

0.6_+0.2 0.64-0.2 10.5:t:0.6 2.5-+0.3 16.6-+0,9 2.0± 0.3 0.7+ 0.2

12.7±0.7 14.4+ 0.8 18.4± 1.0 19.0_+1.0 19.5-+1.0 17.5+ 1.0 6.3_+0.5

The experimentswerecaniedout at 37 °C in 100 mM sodiumphespha~ buffer, pH 7.0, con~alng 6 ltM of zhe lipophilicaed~. ,:~'qlSOcells were addedto a finalcoricencationof 3× 10~ n'd-I. PMB was addedto 150 gg ml-~ andGD to4 gg nd-I. D ~ representmean±S,E. f~"foer to six independentdeterminalkms.

266

R. Daugelaci~ius et al. / Bioelectrochemistry and Bioenergetics 42 (1997) 263-274

Table 2 Effect of lipophilic compounds on the cell viability Co~rgmund

Concentration/ItM

PC'B-

4 46 4000 4 40 4000 4. 40 4000 4 40 4000

NCB-

DPCB-

DCB-

Viability/% Intact E col±

EDTA-treated E col±

B. subtilis

100 99 7 100 99 2 i00 99 0 100 98 I

96 85 6 98 9 2 85 8,5 0 89 8.5 2

97 74 3 100 1I 0 82 0 0 79 0 0

The experiments were carried out at 37 °C in 100 mM sodium phosphate buffer, pH 7.0, containing appropriate concentrations of the LPAs. The intact E col± ANI80 ceils. ANIS0 cells treated with "Iris and EDTA and B. subtilis cells were added to a final concentration of 1 x 10~ ml -I , incubated for 10 rain, diluted and plated. 100% viability corresponds to the amount of colonies in control measurements when the cells were incubated in the buffer without LPAs.

binding. The addition of chloroform to a suspension of E col± cells with permeable OM does not increase the binding of PCB- significantly, although it induces an efflux of accumulated TPP + ions very effectively (not shown).

and/or size helps to bind larger amounts to intact and inactivated cells. The viability studies show that DPCBand DCB- have a higher cytotoxic effect compared with the other LPAs tested (Table 2). However, the intact OM protects E, col± cells rather effectively from the cytotoxic action of DPCB-, DCB- and NCB- in the concentration range 4-40 ixM. At concentrations higher than 40 ItM, all the LPAs studied are highly cytotoxic. In addition, DCBinduces a considerable decrease in the turbidity of bacterial suspensions when its concentration exceeds 1 mM (not shown).

3.2. Binding and cytotoxici~.' depend on the chemical nature of the LPAs In order to examine the cause of the exceptionally low I~CB- binding to intact cells, analogues of this anion were studied. Experiments demonstrate (Table 1) that E. col± cells bind large amounts of DF'CB- and DCB-, but the binding of other PCB- analogues and TPB- is qualitatively similar to that of PCB-. In all cases studied, the PMB/GD-inactivated cells bind larger amounts than the intact cells, More bulky analogues are bound in 15%-55% larger amounts when compared with PCB-, but the amount of bound TPB- is about 50% lower (,Table 1). These results show that an increase in LPA lipophilicity

3.3. PMB, but not heating, considerably increases the binding of LPAs to membrane-containing viruses It is evident that inactivated microbial cells and membrane vesicles, but not intact cells, are able to bind le,rge amounts of PCB- (see Fig. I). In another series of experiments, we used two spherical lipid-containing bacterial

Table 3 Binding of lipophilic anions by membrane-containing bacteriophages and pure phospholipid.,made vesicles ~

Object, amount

Amount of LPA bound/nmol In the presence of PMB

Without PMB

I:'CBBacteriophages PRDI. 2.5 × 10j-" 2,9 ± 0.3 t~6, 3 x 10II 2,8±0.3 lrrmspholipids PE, 10 p.g 2,4 ± 0.3 P(3, 45 ~g 1.2+0.1 CL 45 I.tg 1.0±0,1 Mixture ~', 100pg 13.1 5:0,8

DCB9.3 ± 0.4 15,4±0.9

DPCB-

NCB-

TPB-

7,9+0.3 16,0±0.9

ND 5.9±03

1.4±0.3 1.5±0.1

18.7 ± 1.0 17,5±1.0 9.8±0.5 11,1±0.6 16.0±0.9 16,2±0.9 ND ND

3,1±0.3 2.6±0.3 2,4±0.2 ND

1.9+0.2 <0,3 <0.3 ND

PCB-

DCB-

DPCB-

NCB-

TPB-

19.6±1.0 17.1±0.9

18,5±1.0 17.6+0.9

ND 11,3±0.6

5.1::[:0.3 4,3±0.2

6.5±0.4 19.7±1.1 10.7±0.6 20.0±1.1 6.8±0.4 21.6±1.1 19.5 ± 1.0 ND

19.0+1.1 21.9±1.1 23.9±1.2 ND

7.9±0.5 15.1±0.8 17.1±0,9 ND

5.5±0.4 5.1±0.4 4.8±0.3 ND

7.7±0.3 6.0±0.3

a The experiments were carried oat at 37 °C in 100 mM sodium phosphate buffer, pH 7,0, containing 6 IzM of appropriate LPA. PMB was added to a final concentration of 120 I.tg ml- m.Data represent mean + S,E, for four to six independent determinations. b 75% PE, 20% PG and 5% CL.

R. Daugelavi[i~ et aL/ Bioelectrochemistryand Bioenergetics42 f 1997) 263-274

267

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Iog[PCB"]~ Fig. 2. Effectof the initialconcentrationon the amountof PCB- boundto intactand heat-inactivatedS. cere~'isiae(A), 8, subtilis (B) and E co//ANI80 (C) cells. The experimentswerecarriedout at 37 °C in 100 mM sodiumphosphatebuffer,pH 7.0, containingan appropriateinitialc o r . e e ~ of I~'B-, The concentrationsof the cells in the suspension(cell ml- ' ) were: S. cerecisiae,2.8 × 10r; B. subtilis, 9 × 10g; E. coli, 12 × !0~; 1, P~-i~mcti~a~l cells; 2, intactcells. viruses to probe the binding of LPAs. Bacteriophage PRD1 has an internal membrane that follows the inner surface of the icosahedml protein capsid [33]. In the case of bacteriophage d~6, the membrane surrounds the viral nucleocapsid [351. The membrane-containing viruses PRD1 and ~b6 (Table 3), but not the non-lipid-containing virus T4 (not shown), bind rather large amounts of LPAs. As in the case of cells, DCB- and DPCB- are bound in considerably larger amounts than PCB-. Heated phage PRDI particles bind the same amount of PCB- as intact particles, although there is a 17% + 3% heat-induced increase in the binding of this anion in the case of phage d~6, The addition of PMB induces a considerab!e increase in the amount of PCB- bound by both membrane-containing bacteriophages (Table 3).

bilayer of the cellular and viral membranes. In order to examine the role of lipids in the binding of LPAs to cells and bacteriophages, the lipid/water partition behaviotrx of LPAs was determined using pure phospi-~ipid-made vesicles. The zwitterionic phospbulipid PE and anionic pholipids PG and CL are the major lipids of E. coli cells [42], as well as of the membrane-containing b,~cteriopheges [34,37]. Injection of ethanol-dissolved PE into the buffer decreases the aqueous phase concentralion of LPAs very effectively (Table 3). However, in similar experimeras, PG and CL bind PCB- weakly. The addition of PMB to suspensions of anionic phospbolipid-made vesicles a considerable additional binding of FEB- in all cases studied (Table 3). As in the case of cells and membranecontaining phage particles, the binding of DCBDPCB- to pure lipid-made vesicles is stronger than the binding of PCB- (Table 3).

3.4. Binding of LPAs to pure lipid vesicles depends on the phospholipid used

3.5. The bound LPAs induce changes in the binding capacity of the membranes

On the basis of studies of LPAs using plane lipid membranes and liposomes [11-14], it is expected that these anions will be accumulated in the phospholipid

When different concentrations of LPAs are present in the buffer, the amount bound per added cells varies lin-

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R. Daugelm,iUus et at./BioelectrochemistQ" and Bioenergetics 42 (] 997) 263-274

early with the initial concentration of the anion; the logarithm of the amount of PCB- bound is directly propor-

coil ceils, a sharp increase in the binding of DPCB- at close to 10 -~ M concentration is also observed (Fig.

in the medium for both intact and heat-inactivated cells (Fig. 2). The uptake shows no saturation, even at 4 mM initial concentration. Some deviations from linearity in the binding of PCB- are observed at 10-5--5 × 10 -4 M concentrations in the case of heat-inactivated B. subtilis cells and at somewhat higher concentrations in the case of E. coli cells (Fig. 2(B) and Fig. 2(0). A clear difference in the amount of PCB- bound between intact and heat-inactivated cells is observed at all concentrations measured. However, at 37 °C this difference for S. cerecisiae cells is smaller than at 25 °C (compare Fig. I(A) and Fig. 2(A)). The experiments with DPCB- show that intact and heat-inactivated B. subtilis cells bind almost equal amounts of this more bulky anion (Fig. 3(A)). However, there is a considerable difference in the amount of DPCB- bound between intact and heat-inactivated E. colt cells. Using E.

Fig. 4(A) and Fig. 4(B) show the results of PCBbinding plotted as a double-reciprocal plot (1/amount bound vs. I/concentration free) at four different concentrations of B. subtilis and E. colt cells. The plots meet at the origin, indicating that at high LPA concentrations the binding can be described as partitioning. However, several considerable dr iations from linearity are observed, indicating changes in the mode of binding when the amount of bound PCB- is increased. Fig. 4(C) and Fig. 4(D) show the same data re-plotted in the form of (amount PCB~,,d/[PCB-]ft~e) vs. amount PCB~oun ~. The (amount PCB~,~,j[PCB-]f,,~) ratio decreases as the amount of PCB~oona increases, but an inLease in this ratio is observed during deviations from linearity in the binding (see Fig. 2(B) and Fig. 2(C)). These results indicate that the binding of LPAs to the

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R. Daugelaci~ius et al. / BioelectrochemL ~r~ "and Bioenergetics 42 (1997) 263-274

cells is characterized by a very high capacity. The absorption of more than 300 nmoi of PCB- to 109 heat-inactivated E. coil or B. subtilis cells at 4 mM initial concentration corresponds to the binding of over five molecules of the anion per phospholipid molecule. The accumulation of large amounts of LPAs in cellular membranes is probably the principal reason for the cytotoxic effect of these compounds at concentrations higher than 40 p,M (Table 2).

3.6. The apparent paniti.,n coefficients of LPAs between biological membranes and aqueous medium are ve~ high The experiments shown in Fig. 2 indicate that the binding of LPAs to the cells can be described as the partitioning of LPAs between the cells and the buffer. Partition coefficients allow the binding affinities of membranes of different origins to be compared. However, it is necessary to know the intramembrane concentrations of LPAs for such calculations. The total cytosol (internal solvent) volume of the cells is at least 150 times smaller than the external incubation buffer volume in our measurements, and therefore the PM depolarization-induced increase in LPA concentration in the cytosol has only a minor contribution to the overall inactivation-induced binding of LPAs to the cells. Being even smaller than the cytosol, the intermembrane space (IS) of E. coil is occupied by anionic compounds which are the cause of a considerable Donnan potential (cell wall negative [43]). Therefore the role of IS in LPA binding should also be minor. On the basis of these considerations, the cellular membranes can be regarded as the exclusive region of accumulation of LPAs. Using the data presented above, the partition coefficients of LPAs between the external medium and the membranes were calculated (Table; 4). These coefficients are linearly related to the ratio (amount PCB~o,,,J[PCB-]free). A complex dependence of (amount

269

PCB~o,,.d/[PCB-]f~:~) on the amount of P C B ~ (Fig. 3(C) and Fig. 3(I))) is an indication that the pa~6on coefficients are dependent an the concenwafion of LPAs in the medium and can be considered only as "apparel". In spite of this, the coefficients of pure lipid ves/ck's for various LPAs, determined using different p h o s p ~ are rather close to the coefficients for the same determined using the cells or bacter;a~phages. In ~ , the partition coefficients for PCB- of the membrane vesicles are in good agreement with the coefficiergs of the heat-inactivated cells. These data confirm our assampaon that the membranes are the major binding compartng~ for PCB-.

3.7. Decrease. but not screening, of the surface charges increases the LPA binding Although the binding of LPAs to the membranes can be described as partitioning, the amount of anion bound to the membrane surface is directly related to the imerfac~, rather than the bulk, aqueous cortcenlrafion. Surfaces of probably all microbial cells are anionic [44] and are at a negative electrical potential with respect to the b u r aqueous phase at physiological pH. The G o u y - ~ theory of the diffuse double layer [13,45] predicts that the concentration of monovalent anions at the surface of the membraue will be an order of magnitude lower than the cancer tration of these anions in the bulk aqueous p!mse when 25% of the membrane lipids are anionic and the concenwation of monovalent cations in the solution is 100 raM. Therefore the surface charge could be one of the factors preventing the binding of LPAs to the mon,~'aaes. To study the effect of surface charge on the binding of LPAs, cells, virus particles and lipid vesicles were incabated in buffers containing different conconWatioas of salts (Table 5). Salt additions decrease the magnitude of the surface potential (~o) by screening the surface charges without changes in the charge density. However, at con-.

Table 4 Lipid/water partition coefficients of PCB- a

Object

Partition coefficient

Heat-inactivated E. coli ANI$0 cells Heat-treated membrane vesicles of E. coil AN iS0 Bacteriophages PRD I

45600 22600

63500 43100

,t,6

14400 55900

46500 136000

Phospholipids PE I'(3 CL Mixture ~'

440OO 4700 40OO 39200

139800 62200 32900 938OO

In the absence of PMB

in the presence of PMB

The data for the calculations of the partition coefficients are taken from Fig. 1 (E. coil heat-inactivated cells and vesicles plus data of the E~,IB effects (not shown)) and Table 3, b As in Table 3.

R Daugelari~ius et aL / gioelectrochemisto" and Bioenergetics 42 (1997) 263-274

270

Table 5 Effects of salts arid temperature on PCB- binding by cells, bacteriophages and vesicles Object

Buffer

Amount bound Without PMB

KO1489

E. coli vesicles PRDI

NaPi, 100 raM, pH 7.0 +1 mM CaCl_~ + 10 mM MgCI, Tris/HCl. 10 raM, pH 7.0 + 10 mM MgCI., NaPi. I00 raM, pH 7.0 Tris/HCl. I00 mM. pH 8,0 + 300 mM NaCI + 2 mM MgCI., + 10 mM MgCI z + 100 mM MgCI 2 NaPi, 100 raM, pH 7.0 + 10 mM MgCI 2 Tfis/HCI, 100 raM, pH 7,0 + I mM MgCl 2 + 10 mM MgCI z + 100 mM MgCI 2 + 1 mM CaCI, + 10 mM CaCI, + 1O0 mM CaCI.,

in the presence of PMB

24 °C

37 °C

24 °C

37 °C

5.8 5:0.5 8.2+0.6 9.7 + 0.6 6.5 + 0.5 9.0 + 0.6 2.7 + 0.3 2.4 + 0.2 2.5 + 0.2 2.7+0,3 2.9 + 0.2 4.2 + 0.3 3.5 + 0.4 6.8 ± 0.4 1.8 5:0.3 3.9 + 0.2 4.2 + 0.3 4.4 + 0.3 2.3 + 0.2 5.0 5:0.3 5.1 5:0.4

8.8 4- 0.4 10,0+0.6 I i, 1 _+0.6 ND ND 2.5 + 0.3 2 0 4- 0.2 2.3 + 0.3 2,2±0.2 2.5 4- 0.2 2.6 + 0.2 2.3 + 0.2 4.4 5:0,2 2.3 5:0.2 3.7 4- 0.2 3.9 4- 0.3 4. I 4- 0.2 2.1 + 0.1 3.9 5:0.2 4.4 4- 0.2

23.0 + I.I 22.6+ 1.1 23.5 + I. I 12.8 -t-0.8 t2.2 + 0.8 7.7 5:0.5 9,1 + 0.5 6.8 :t: 0.4 9.1 +0.5 5.8 + 0,4 6.8 + 0,4 17.7 5:0,9 18.3 4- 1,0 17.6 5:1.0 15,1 -t- 0.8 13.2 + 0.6 ! 1.7 + 0.6 14.5 4- 0.6 13.0 5:0.7 9.7 5:0.5

21.2 4- !.1 21.8+ i.0 2 i,6 + 1.0 biD ND 7.4 5:0.4 8.4 4- 0.6 6.2 4- 0.5 8.3 +0.4 5,4 + 0.4 4.3 + 0.3 13,7 5:0.6 13.9 5:0.6 17.5 + 1.0 14.1 + 0.7 12,5 5:0.6 9.2 + 0.5 13.3 ff 0.6 10,6 5:0,5 8,9 5:0,4

The experiments were camed out il. 5 ml of a buffer containing 6 o,M of PCB-. E. coil KO1489 cells were added to a final concenuation of 3 × 109 cells ml -t. E. coil ANI80 vesicles to 4( i,tg of protein ml -~. phage PRDI to 5 x l0 N particles ml -I and PG to 20 p,g ml -I, PMB was added to a final concentration of t20 p.g ml-n. Data represent mean + S.E. for tour independent determinations. NaPi indicates sodium phosphate buffer.

centrations up to 300 mM, NaC! affects the binding of LPAs rather weakly. Divalent cations reduce ~'0 more effectively than monovalent cations. It is known [13] that, in the presence of 10 mM MgCl 2 in 100 mM sodium phosphate, the surface potential of biological membranes is abolished. However, the results in Table 5 show that the binding of LPAs increases onl,, slightly when the Mg 2+ or Ca-'+ concentration is increased from 1 to I00 mM. The small effect of salts on PCB- binding indicates that the

30--~---~m--~-~--.~

screening of the external surface charge is not sufficient to cause strong binding of the LPAs to membranes. However, the PMB effect on the binding of LPAs is noticeably blocked by high concentrations of divalent cations. The measurements of PCB- binding were performed at two temperatures (24 °C and 37 °C, Table 5). A small, but reproducible, decrease in the amount of PCB- bound is recorded at the higher temperature. This is in agreement with the results obtained with artificial membranes [14]. 25

i"

A

E

2O 2O 0 -Q

15

\

~5

10

°~\"O t

"\

5 o

~

<

o

2

3

4

5

6

7

8

"0 2

ol

1

I

~

N

l

f

l

l

2 3 4 5 6 7 8

pH Fig. 5. Effect of pH on the amount of bound PCB- by E. coil KO1489 cells, bacteriophage PRDI and stabilized olive oil emulsion (SOOE). The experiments were carried out at 24 °C in 30 mM citric acid/50 mM NaCI/HCI (pH 2), 50 mM citric acid/NaOH (pH 3-6) or 100 mid sodium phosphate buffer (pH 6.0-8.0), containing 6 I~M of PCB-. E. coil cells were added to a final concentration of 3 × l09 cells ml- i PRDI to 5 × 10 tt particles ml- i and l : ! (vol/vol) SOOE to 51,1,1of olive oil ml-J. PMB was added to a final concentration of 120 p,g ml- ~. A: curve 1, E. coli ia the presence of PMB; curve 2, E. coil without PMB; curve 3, PRDI in the presence of PMB; curve 4, PRDI without PMB. B: curve l, SOOE in the presence of PMB; 2, SOOE without PMB.

R. Daugelavi~iuset al,/ Bioelectrochemistryand Bioenergetics42 (1997~263-.274 The pH dependence of the amount of LPAs bound was also studied. A decrease in pH makes ~o more positive by inducing the protonation of groups of a dissociative nature in phospholipids. The pH effect on the binding of LPAs to cells, membrane-containing phage particles and lipid vesicles is obvious (Fig. 5(A)). In all cases studied, larger amounts of LPAs are bound when [H +] is increased. This effect is particularly well expressed below pH 4.

3.8. Lipophilicity-dependent binding of the LPAs The lipophilicity of a compound is generally deduced from the partition coefficient between water and octanol or olive oil. These coefficients have been found to have a rather good correlation with data on a wide range of physiological effects of lipophilic compounds [46]. The accumulation of LPAs by cells is likely to occur on the surface of phospholipid bilayers, and therefore a biological raembrane can be considered not as a bulk but as an anisotropic interfacial phase. The question arises as to what extent the effects registered are determined by the hydrophobicity and/or by the unique structural features of biological membranes. In order to distinguish the bilayer effects from the membrane hydrophobic phase effects, the partitioning of LPAs into the bulk phase of organic solvents was determined. Experiments with octanol and olive oil show that the amounts of LPAs bound depend notably on the form of the organic solvent studied (Table 6). Small volumes of pure octanol ¢r olive oil injected into aqueous buffer bind considerably lower amounts of PCB- when compared with the case in which the same amounts of these solvents are injected as 1:1 (voi/vol) solutions in ethanol. The binding of LPAs by stabilized olive oil emulsion (SOOE)

Table 6 Organic phase/water partition coefficientsof lipophilicanions Substance Anion Partitioncoefficient Without PMB

Oliveoil Oliveoil:ethanol Octanol Octaaol:ethanol SOOE Oliveoil Octanol SOOE SOOE SOOE SOOE

PCB-

DPCBDCBNCBTPB-

271

is the highest. This dependence on the form of the o r g ~ solvent used probably has a kinetic naa~re. It is possib~e that the stabilization of the LPA concentration in medium indicates only the saturation of the surface lay¢: of the organic phase. However, even in the case of SOOE, the organic phase/buffer Fartition coefficionts for PCBare more than two orders of magnitude lower than the apparent membrane/buffer partition coefficients (Table 4). The results of these experiments also confirm onr assumption that DPCB- and DCB- are more lipophilic hhan the other LPAs studied (Table 6). The addition of PMB increases the amount of LPAs bound to the organic phase, although its effect on the strong binding of DPCB- and DCB- is small. As in the case of the membranes, the amount of ~ bound by SOOE is higher at low pH (Fig. ~(B)). However, the dependence of the amount bound on pH is different from that observed for the membranes. In the presence of PMB, the amount of PCB- bound to SOOE exhi~ts a maximum at pH 5. These results indicate that the hireling stimulating activity of PMB is also pH depeadem, In absence of PMB, a rather even increase in PCB- binding to SOOE with decreasing pH is observed.

4. Discussion Two categories of LPA binding regulating factors can be defined: (I) intrinsic factors affecting LPA binding to biological and artificial membranes; (2) factors preventing LPA binding to the PM of intact cells, These factors can affect the binding capacity of the membranes and/or form a barrier to the access of the membrane in~erior. According to the bilayer couple theory [47], membranes in which polar lipids are distributed asymmetrically can act as hilayer couples. In such a case, a low LPA bh~ling capacity of the outer phospbolipid layer of the cellular membrane can also be considered as a barrier to anions to access the inner membrane.

With PMB

15 35 20

4.1, Intrinsic factors affecting LPA binding to biological and artificial membranes

55

it is obvious that the dipole potential, produced by molecular dipoles at membrar~[solution interfaces [19], is a main factor determining the LPA binding cap~ity of pbospholipid monolayers and bilayers [12,18]. It is also known [48] that an increase in the membrane surface density leads to an exclusion of lipophilic solutes. It is no~ known how strongly the latter factor influences the binding of LPAs, which are accumulated not at the centre of the bilayer, but at the surface regions. However, a gel-to-fluid phase transition in dipalmitoylphosphatidyicholin~, membranes is followed by a transition from a weakly to strongly LPA-adsorbing state [11]. The decrease in the ratio (amount PCB~,ad/[PCB-]f~)

< 10 20 10 30 90 40 75 310 36O 220

250 140 490 550 62O 410

40

85

The experimentswerecarriedout at 24 ~Cin 100 mM ~uxliumphosphate buffer, pH 7.0, 50-150 itl of pure octanol of olive oil, I : 1 (vol/'voi) solutionsof these substancesin ethanol or I : ! (vol/vol) stabilizedolive oil emulsion(SOOE)in waterwere injectedinto the buffet.The coefficients were calculatedfrom the amount of LPA bound by the organic phase. The initialconcentrationof LPAswas 6 ttM. PMBwas addedto a final concentrationof 120 ttg ml"°.

272

R. Da,~gelavi~iuset al. / Bioelectrochemist~'and Bioenergetics42 {I997) 263-274

with an increase in the amount of anion bound to heat-inactivated cells (Fig. 4(C) and Fig. 4(D)) indicates a saturation process in the binding of LPAs to biological membranes based on the charge and/or volume of the anions bound. A temporary, but considerable, increase in this ratio at LE a. concentrations above 10 -5 M indicates autostimulafion of the binding. This may be due to the LPA accumulafion-indnced expansion of the membrane surface [49,50] and/or formation of new membrane vesicles [51]. As a consequence of the increased area of the bilayer and/or decrease in the surface density, the binding capacity of the membranes increases. This sharp increase in the binding capacity of the membranes indicates an abrupt stage in the process of membrane disruption by LPAs probably causing an h-reversible loss of the viability of the cells (Table 2). Similar anomalies in ~ potential isotherms registered in the same interval of LPA concentrations can be explained by lipophilic ion-induced isothermal transitions of membranes from the gel to the fluid state [1 !]. The overall phospholipid bilayer of bacterial membranes is in the fluid state at 37 °C [42]. However, the coexistence of gel and fluid domains is l~sible in bacterial membranes, and LPAs may induce a phase transition in the former. The increase in the binding of LPAs to membrane-con~ning bacteriophages, pure phospholipids and E. coli-derived membrane vesicles, induced by high concentrations of divalent cations (Table 4), can be explained by a destruction of the electrostatic barrier to anions at the membrane surface. However, the salts only screen the negative surface charge. The reason for the considerable difference between the effects of salt and pH is that a decrease in pH across the isoelectric pH of the membrane surface (usually in the pH range 4-5 [45]) changes the sign of the surface charge. A positive membrane surface charge at low pH is prebably the cause of the increased LPA binding to all types of membranes studied. However, in the case of bacteria, a declease in viability is also observed at pH < 3 (not shown). Amphipathic polycationic antibiotic PMB increases the amount of LPAs bound by all types of membranes and organic solvents studied. Due to its structural features, PMB probably concentrates at the interfacial region and decreases the energetic barrier for LPAs to enter the organic phase. Insertion of this bipolar molecule disrupts the barrier to LPAs, converting the negative bilayer surface charge to a positive charge. In addition, PMB can also affect the dipole potential and the surface density of the membranes. 4.2. Factors preventing LPA binding to the PM of intact ceils

The lower binding of LPAs to intact ceils may be caused by a metabolism-induced decrease in the binding capacity of the PM. However, the relation of the dipole potential as well as the membrane surface density to the cellular metabolism is unclear. Bacteriophage T4 increases

the binding of PUB- to intact E. coil cells very effectively (Fig. I(C)), although at low multiplicities it does not considerably affect the metabolism of the cells [52]. it is known that phage T4 induces fusion zones between the PM and the OM [53] and forms ion-permeable channels in the envelope of E. coli cells [26,40]. Therefore it is probable that the reduced binding of LPAs is due to difficulties of partitioning into the membranes of intact cells. The formation of a barrier to LPAs in intact microbial cells can take place at a number of stages, and there may be a combination of causes for exclusion. The following exclusion systems may operate: (1) structural and electrostatic barriers of the cell wall, including the LPS-dependent OM barrier of the Gram-negative cells; (2) PM-associated metabolically developed barriers, including envelope-ass¢:ciated energy-driven pumps, extruding molecules that have already crossed other barriers. It i: known (see Ref. [54]) that the permeability of the OM depends on the metabolic state of the cell. As cells with a permeable or permeabilized OM bind more PUBthan cells with an intact OM (Fig. I(C)), it is evident that the outer layer of the OM contributes in preventing LPA binding and the OM can bind LPAs after destabilization of its structure. Binding of about one-third of the maximum PCB- amount bound to cells with permeable OM is in agreement with the results of Vaara et al. [55], which indicate that the lipopolysaccharide/water partition coefficients of various hydrophobic probes are about one order of magnitude smaller than the phospholipid/water partition coefficients. Therefore it is possible to consider the envelope of Gram-negative bacteria as a system with three PUB- binding zones: the inner layer of the OM and both layers of the PM. Stronger binding of the very lipophilic anions DPCB- and DCB- to the outer leaflet of the OM may be one of the reasons why E. coli cells with an intact OM are not killed by these anions at 4-40 I~M initial concentrations despite strong binding (see Tables I and 2). Metabolism-inhibiting agents deplete the cell wall of H + and consequently expose an increased number of negatively charged groups. As a consequence, a smaller amount of anions should be bound to the cell. This has been observed with chromate [56] which does not penetrate into the cell. However, the opposite effect is demonstrated here with PCB-. This emphasizes that LPAs differ in nature from inorganic anions and are accumulated in the cells by very different mechanisms. The surface area of the peptidoglycan sacculus depends on the metabolic state of the cell [57]. However, it is not probable that the peptidoglycan layer plays a role as a structural barrier to LPAs as its porosity in growing cells is considered to be greater than after inactivation. We have shown that the OM of Gram-negative bacteria is not the sole factor preventing the access of lipophilic compounds to microbial cells, and that energy transduetion plays an important role in preventing LPA binding. This

R. Daugelovi~iuset al. / Bioelectrochemistryand Bioenergetics42 (I997) 263-274

locates an additional barrier function to the metabolically active PM. The best known mechanism for preventing the access of lipophilic compounds to the membrane interior is the membrane-associamd energy-driven efflux (for a review, see Ref, [58]). Bacterial efflux sysmms of extremely broad substrate specificity play a major role in drug resistance, especially in combination with the permeability ba,~er. Howcver, it seems unlikely that the active pumping of LPAs from the cellular membranes is a major contributor to the barrier function. The pumps alone would not be able to ensure a considerable difference in the binding at l0 -4 M and higher concentrations of lipophilic compounds [59]. It is possible that only the outer leaflet of the PM is involved in the barrier function in intact cells. PG- or CL-made vesicles bind a small amount of PCB-, bnt a large amount of DCB- or DPCB- (Table 3). Therefore the PCB- binding capacity of the oumr layer of the PM should be low because of the large amount of negatively charged phospholipids [60]. The inner surface of the PM is rich in PE, which actively binds LPAs (Table 3). However, it is unattainable to LPAs in intact cells. Phage 1"4 induces the effective binding of LPAs to infected E. coil cells by fomfing fusion zones between the OM and the PM. In such a case, the anions penetrate directly from the incubation medium to the region of strongest binding (inner leaflet of the PM), avoiding the OM, intermembrane space and contact with the barrier located at the outer surface of the PM. We conclude that LPA binding to microbial cells is regulated by intrinsic membrane as well as metabolism-dependent factors. Besides the well-known dependence on the iipophilicity of the anion and temperature of the medium, the amount of LPA bound also depends on the phospholipid composition of the membrane, pH and ionic composition of the incubation medium. We have also demonstrated that the PM, in addition to the OM, of Gram-negative bacteria creaks an effective barrier for LPAs.

Acknowledgements Ms. Jolanta Kulakauskien6 and Ms. Marja-Leena Peral~i are acknowledged for their skilful technical assistance. R.D. was a recipient of a scholarship from the Nordic Council of Ministers to work at the Division of Genetics, University of Helsinki,The support of the Finnish Academy of Sciences to D.H.B. is gratefully acknowledged. This investigation was also supported in part by the EC-SCIENCE grant SCI *-CT91-0735.

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