386
Biochimica et Biophysica 4cta, 539 (1978) 386--397 © Elsevier/North-Holland Biomedical Press
BBA 28453 E F F E C T S OF STAPHYLOCOCCIN 1580 ON CELLS AND MEMBRANE VESICLES OF B A C I L L U S S U B T I L I S W23
A. WEERKAMP * and G.D. VOGELS Department of Microbiology, Faculty of Science, University of Ni]megen, Ni]megen (The Netherlands)
(Received August 16th, 1977)
Summary 1. Uptake o f L-glutamic acid is inhibited, and preaccumulated L-glutamic acid is released from Bacillus subtilis cells treated with staphylococcin 1580. Uptake of a-methylglycoside is enhanced at low bacteriocin concentrations and inhibited by excess bacteriocin. 2. Inhibition of amino acid uptake into m e m b r a n e vesicles is somewhat less sensitive to staphylococcin 1580 than uptake into whole cells under similar conditions, when the bacteriocin c o n c e n t r a t i o n is expressed per weight unit of m e m br an e protein. Inhibition of uptake into vesicles is i n d e p e n d e n t of the electron d o n o r system used, but varies with different substrates. 3. Influx of L-glutamic acid into vesicles under anaerobic conditions is severely hampered by staphylococcin 1580. The L-glutamic acid carrier functions are slightly affected only. 4. Scaphylococcin 1580 abolished the m e m b r a n e potential induced by respiration or by a potassium diffusion potential in the presence of valinomycin, as measured with the fluorescent dye 3,3'-dipropylthiadicarbocyanine. 5. The effects of staphylococcin 1580 on cells and m e m b r a n e vesicles allowed the classification into three groups with different sensitivity to the staphylococcin concentration.
Introduction Bacteriocins are protein antibiotics, which are bactericidal to bacteria related to the producing strain. One class of bacteriocins is assumed to exert its action directly on the cytoplasmic membrane. Among them, especially colicins E1 and
* To whom correspondence should be directed at the present address: Department University of British Columbia, Vancouver B.C. V6T 1W5, Canada.
of Microbiology,
387 K [1] have been studied extensively. Also staphylococcin 1580, a bacteriocin p r o d u c e d by Staphylococcus epidermidis, has been studied to considerable depth [2--5]. Like colicins E1 and K, staphylococcin 1580 rapidly induces an inhibition of macromolecular syntheses and active transport, depletion of cellular ATP and leak of preaccumulated Rb ÷ from the cells [2]. However, the permeability to p r o t ons is n o t affected [3,5]. Sensitivity of cells to staphylococcin 1580 is d e p e n d e n t on the physiological state of the cells, and is influenced by conditions such as the t em perat ure during growth and incubation, energy charge of the cells and the kind of carbon source applied in the medium [5]. It was suggested that the primary effect of staphylococcin 1580 is the uncoupling of the m em brane potential and electron transport [5]. In spite of the advantages m e m b r a n e vesicles offer to study energy coupling and transport p h e n o m e n a , very few reports considered the effects of bacteriocins on vesicles [3,6]. J e t t e n and Vogels [3] showed that transport of amino acids in memb r an e vesicles was inhibited by staphylococcin 1580. This study aims to compare the action of staphylococcin 1580 on whole cells and membrane vesicles, and to e xt e nd the previous experiments as to the m ode of action o f the bacteriocin. In the present study Bacillus subtilis W23, an organism equally sensitive to staphylococcin 1580 as the previously used Staphylococcus aureus [3] was used as the indicator strain. Uptake of various substrates into vesicles prepared from B. subtilis W23 has been extensively studied by Konings et al. [7--9]. T h e y showed that these vesicles are essentially right side out and t hat transport rates of vesicles appr oxi m a t e those of intact cells. Materials and Methods
Bacterial strain, medium and growth conditions. B. subtilis W23 was grown in nutrient-sporulation medium [10] at 37°C with vigorous aeration. The medium was inoculated with an overnight grown culture to obtain an absorbance at 600 nm (A600nm) of 0.05, and harvested in mid-exponential phase (A600n m 1.2). The cells were washed once with 0.1 M potassium phosphate (pH 7.3), and centrifuged 10 min at 12 000 × g. Preparation of membrane vesicles. Membrane vesicles were prepared by direct lysis in h y p o t o n i c medium, as described by Konings and coworkers [7,11], with the e x c e p t i o n t hat the cells were harvested in the mid-exponential rather tt, n in the early stationary phase. Sodium-loaded vesicles were prepared by an identical procedure, e x c e p t that sodium buffers replaced potassium buffers t h r o u g h o u t the procedure. Vesicles were rapidly frozen in thinwalled plastic tubes by the use of liquid nitrogen and stored a t - - 8 0 ° C . Immediately before use vesicles were rapidly thawed at 46°C in a water bath; only vesicles th at were frozen once were used. Transport experiments. Incubation mixtures contained, unless otherwise indicated, final concentrations of either 50 mM potassium or sodium phosphate (pH 6.6), 10 mM MgSO4 and 0.2--0.5 mg of m e m b r a n e protein per ml in a total volume o f 50--250 gl. Mixtures were incubated at 25°C with a stream of water-
388 saturated oxygen blowing over the surface. After 2 min preincubation the electron d o n o r was added, followed I min later by the labelled substrate. Initial uptake rates were measured after 30 s by terminating the reaction by the addition o f 2 ml 0.1 M LiC1, as described earlier [3]. The time course of uptake was measured by removing samples from the incubation m i xt ure at various time intervals with an O x f o r d sampler (Oxford Lab. Intern. Corp.). Samples were spotted on a p r e w e t t e d 0.45 gm pore size m e m b r a n e filter (Millipore Corp.) and washed with 3 ml 0.1 M LiC1, at r o o m temperature. Uptake experiments in whole cells (about 1.2 mg dry weight per ml) were p e rf o r med as described for m e m b r a n e vesicles. Fluorescence measurements. Fluorescence of 3,3'-dipropylthiadicarbocyanine was used to m o n i t o r the m em br ane potential [12,13] and was carried out as described previously [ 5]. The incubation mixtures (2.5 ml final volume) contained 50 mM of either sodium or potassium phosphate (pH 6.6), 10 mM MgSO4 and 20 pg m e m b r a n e protein per ml. 3,3'-Dipropylthiadicarbocyanine was added to a c o n c e n t r a t i o n of 0.8 ~M and fluorescence was excited at 620 nm and measured at 662 nm, in an Aminco Bowman spectrofluorimeter. Measurements were carried out at 25°C. The percent fluorescence quenched was calibrated with a potassium diffusion potential to estimate the respirationinduced memb r an e potential. Potassium-loaded vesicles were diluted into phosphate buffers containing different p r o p o r t i o n s of sodium and potassium phosphate. The fluorescence quench occurring u pon addition of valinomycin was recorded and p lo tt e d against the diffusion potential, calculated from the Nernst equation (A~ = 59 log K~n/Kout). Our results were similar to those obtained by B h a t t a c h a r r y y a et al. [12] who used Azotobacter vinelandii vesicles. Preparation and assay o f staphylococcin 1580. Product i on and purification o f the bacteriocin were described previously [4]. Briefly, the bacteriocin was isolated from supernatants of S. epidermidis 1580 cultures in medium A with 1% sodium pyruvate, and purified by a m m o n i u m sulfate precipitation and chrom a t o g r a p h y on CM-Sephadex C25. The final preparation was hom ogeneous on polyacrylamide gel electrophoresis and isoelectrofocussing, and usually contained a b o u t 25 000 arbitrary units per mg protein. Activity was measured as described previously [14] and expressed as arbitrary units per ml. Determination o f viability. Cells were incubated at 25°C as described for the uptake experiments. After 2 min of preincubation, staphylococcin 1580 was added and after a not her 4-min samples were removed, rapidly diluted 1000fold in p e p t o n e water (Oxoid Ltd, L o n d o n ) and plated in suitable dilutions on n utr ien t agar (Oxoid) containing 0.2 M NaC1. The results were expressed as percent survivors relative to cell suspensions n o t treated with staphylococcin 1580. Protein determination. Protein was measured according to the m e t h o d of L o w ry et al. [15]. The m em br ane protein c o n t e n t of whole cells was assumed to be 15% of the total protein c o n t e n t [16]. Chemicals. 3,3'-Dipropylthiadicarbocyanine was a gift from A. Waggoner, Amherst College, Amherst, Mass. Nigericin was donat ed by Dr. W.E. Scott, H o f f m a n La Roche, Nutley, N.J. Valinomycin was obtained from Calbiochem, Lucerne, Switzerland; gramicidin D was from Boehringer, Mannheim, G.F.R.,
389 and 5-N-methylphenazonium methosulfate from B.D.H. Chemicals Ltd., Poole, U.K. Radiochemicals, obtained from Radiochemical Centre, Amersham, U.K., were L-[UJ4C]glutamic acid (270 Ci/mol), L-[UJ4C]glutamine (40 Ci/mol), L-[U-14C]leucine (330 Ci/mol), L-[UJ4C]lactate (sodium salt) (50 Ci/mol), [1J4C]acetate (sodium salt) (58 Ci/mol), [3H]inulin (695 Ci/mol), S6RbC1 (2-10 Ci/g), and methyl (a-D-[UJ4C] gluco)pyranoside (3 Ci/mol). Results
Effect o f staphylococcin 1580 on active transport in whole cells and membrane vesicles o f B. su btilis It was previously shown that staphylococcin 1580 blocks active uptake of amino acids in cells [2] and membrane vesicles [3] of S. sureus, and causes the efflux of preaccumulated solute. Fig. 1 shows that uptake of L-glutamate in cells of B. subtilis is inhibited and that the preaccumulated amino acid is rapidly released under the influence of the bacteriocin. However, similar concentrations of staphylococcin 1580 have much less severe effect on the uptake of ~-methylglucoside. Relatively low concentrations of staphylococcin 1580 even enhance uptake significantly, up to a maximal stimulation of 40% under the conditions tested. In B. subtilis ~-methylglucoside is transported by the phosphoenolpyruvate:glucose phosphotransferase system, and retained within the cell both as ~-methylglucoside phosphate and, after dephosphorylation, as ~-methylglucoside, which are not further metabolized under the experimental
UPTAKE[nmoles/mgMEMBRANEPROTEIN] 50f
500
L-GIu
LO
/" / "
30
~MG
LO0 300
/
20
200
10
100 /
/
,
f 2
4
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I
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l. 6 TIME[min]
F i g . 1. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n t h e u p t a k e o f L - g l u t a m a t e ( L - G l u ) a n d ~ - m e t h y l g l u c o s i d e (c~MG) b y cells o f B. s u b t i l i s W 2 3 . Cells w e r e g r o w n i n t o m i d - e x p o n e n t i a l p h a s e , w a s h e d a n d r e s u s p e n d e d in 50 m M p o t a s s i u m p h o s p h a t e ( p H 6 . 6 ) , a n d 10 m M M g S O 4, at a final c o n c e n t r a t i o n o f 1 . 2 m g d r y w e i g h t p e r m l . U P t a k e e x p e r i m e n t s w e r e c a r r i e d o u t a t 2 5 ° C as d e s c r i b e d , b u t an e x o g e n o u s e n e r g y s o u r c e w a s o m i t t e d . S t a p h y l o c o c c i n 1 5 8 0 w a s a d d e d 2.5 r a i n ( i n d i c a t e d b y t h e a r r o w ) a f t e r t h e a d d i t i o n o f t h e label in a final c o n c e n t r a t i o n o f 10 ( o ) a n d 3 5 0 a r b i t r a r y u n i t s p e r m l (A), r e s p e c t i v e l y . C o n t r o l s r e c e i v e d n o s t a p h y l o c o c c i n ( e ) . T h e u p t a k e of L - g l u t a m a t e ( 1 1 . 7 p M ) a n d ~ - m e t h y l g l u c o s i d e ( 1 . 5 r a M ) w a s p l o t t e d against the time.
390
SOLUTE UPTAKE [% of control] - VIABILITY
100 -
6O
2O
1
2
3
L,
LOG AU./rag MEMBRANE PROTEIN Fig. 2. C o r r e l a t i o n b e t w e e n t h e c o n c e n t r a t i o n o f s t a p h y l o c o c c i n 1 5 8 0 a n d i t s e f f e c t o n t h e v i a b i l i t y of c e l l s , o n L - g l u t a m a t e u p t a k e in cells a n d m e m b r a n e vesicles a n d o n ( ~ - m e t h y l g l u c o s i d e u p t a k e in cells. Upt a k e r a t e s o f L - g l u t a m a t e ( 1 1 . 7 p M ) a n d e - m e t h y l g l u c o s d e ( 1 . 5 r a M ) w e r e m e a s u r e d a f t e r 30 s, a n d a m o u n t e d t o 1 2 . 2 a n d 1 1 5 . 0 n m o l / m i n p e r m g m e m b r a n e p r o t e i n , r e s p e c t i v e l y , w h e r e a s 9 . 4 n m o l L-glut a m a t e w e r e t a k e n u p p e r r a i n a n d p e r m g m e m b r a n e p r o t e i n in t e s t s w i t h vesicles. S t a p h y l o e o c c i n 1 5 8 0 , d i s s o l v e d in b u f f e r , w a s a d d e d 1 r a i n p r i o r t o t h e a d d i t i o n o f t h e l a b e l l e d c o m p o u n d ; c o n t r o l s r e c e i v e d o n l y t h e b u f f e r . U p t a k e in m e m b r a n e s vesicles w a s s t i m u l a t e d b y s o d i u m a s c o r b a t e ( 2 0 r a M ) a n d 5-Nm e t h y l p h e n a z o n i u m m e t h o s u l f a t c ( P M S ) (0.1 m M ) ; in e x p e r i m e n t s w i t h w h o l e cells no e x o g e n o u s e n e r g y s o u r c e w a s a d d e d . T h e v i a b i l i t y w a s m e a s u r e d a f t e r t r e a t m e n t o f t h e cells w i t h s t a p h y l o c o c c i n f o r 4 rain. R e s u l t s w e r e e x p r e s s e d as t h e p e r c e n t a g e o f c o n t r o l e x p e r i m e n t s w i t h o u t s t a p h y l o c o c c i n 1 5 8 0 , a n d p l o t t e d as a f u n c t i o n o f t h e s t a p h y l o c o c c i n c o n c e n t r a t i o n . O p e n s y m b o l s : ©, L - g l u t a m a t e u p t a k e , w h o l e cells; A ~ - m e t h y l g l u c o s i d e u p t a k e ; D v i a b i l i t y . C l o s e d s y m b o l s : • L - g l u t a m a t e u p t a k e , m e m b r a n e vesicles.
conditions [17]. The r e t e nt i on of a-methylglucoside within the cells indicates that the integrity of the cell m e m b r a n e is conserved when staphylococcin was applied in concentrations t hat effectively inhibited glutamate uptake. The uptake o f a-methylglucoside was inhibited when higher concentrations o f the bacteriocin were used (Fig. 2). This effect is presumably due to an increase of the permeability of the membrane. Similar results have been reported for Escherichia coli cells treated with colicins E1 and K [18] or Ia [19], except that several-fold stimulations were f o u n d with cells grown on glycerol. The killing o f the cells correlates with the inhibition of L-glutamate uptake (Fig. 2) if the bacteriocin c o n c e n t r a t i o n is expressed as arbitrary units per mg m e m b r a n e protein. This way of plotting was chosen in order to compare the effect o f staphylococcin 1580 on bot h cells and m e m b r a n e vesicles and is based on the assumption t hat the m e m b r a n e is the direct target for the action of staphylococcin 1580. If expressed in this way transport rates for L-gluta-
391
mate were a b o u t similar in whole cells and m e m b r a n e vesicles [11,21], b u t a five times higher staphylococcin 1580 c o n c e n t r a t i o n is required to inhibit t r a n s p o r t in m e m b r a n e vesicles to the same e x t e n t as in whole cells. This difference presumably is n o t the result o f a difference in the a m o u n t of staphylococcin 1580 adsorbed by either cells or vesicles, since whole cells can adsorb more bacteriocin than vesicles can (under the experimental conditions a b o u t 750 and 250 arbitrary units per mg m e m b r a n e protein, respectively). It was previously shown t ha t staphylococcin 1580 has a strong t e n d e n c y to stick aspecifically to both cell walls and membranes of sensitive as well as resistant bacteria [22]. It should be noted, however, t hat the ratio between specific and unspecific adsorption is u n k n o w n . Table I shows the ef f ect of staphylococcin 1580 on the uptake of various c o m p o u n d s by m e m b r a n e vesicles, and the influence of various electron d o n o r systems. The initial upt a ke of L-glutamate was inhibited to the same ext ent , whenever NADH, s ucci nat e/ 5- N - m et hyl phenazoni um methosulfate, or ascorb a t e / 5 - N - m e t h y l p h e n a z o n i u m m e t hos ul f a t e were used as electron donors. This result is conceivable, since staphylococcin 1580 does n o t affect the electron transport, or m e m b r a n e - b o u n d dehydrogenases [3]. The differences observed between the transport inhibition of various substrates by the bacteriocin can-
TABLE I E F F E C T O F S T A P H Y L O C O C C I N 1580 O N T H E U P T A K E O F V A R I O U S S U B S T R A T E S BY MEMB R A N K V E S I C L E S O F B. S U B T I L I S W 2 3 , E N E R G I Z E D B Y V A R I O U S E L E C T R O N D O N O R S P o t a s s i u m - l o a d e d v e s i c l e s ( 0 . 3 7 m g p r o t e i n p e r m l ) w e r e i n c u b a t e d at 2 5 ° C w i t h 50 m M p o t a s s i u m p h o s p h a t e ( p H 6 . 6 ) , a n d 10 m M M g S O 4 in a final v o l u m e o f 55 pl. 2 5 0 a r b i t r a x y u n i t s o f s t a p h y l o c o c c i n 1 5 8 0 ( 6 7 5 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ) w e r e a d d e d i m m e d i a t e l y p r i o r t o t h e a d d i t i o n o f t h e electron donors. A f t e r 1 m i n of i n c u b a t i o n the u p t a k e was started by the addition of the labelled c o m p o u n d s . T h e u p t a k e w a s t e r m i n a t e d 30 s l a t e r a n d t h e r a d i o a c t i v i t y w a s m e a s u r e d . T h e e l e c t r o n d o n o r s u s e d w e r e : N A D H ( 5 raM); s o d i u m a s c o r b a t e ( 2 0 r a M ) a n d 5 - N - m e t h y l p h c n y l a z o n i u m m e t h o s u l f a t e ( P M S ) (0.1 m M ) ; s o d i u m succinate (10 raM) and 5 o N - m e t h y l p h e n a z o n i u m m e t h o s u l f a t e (0.2 raM). Final c o n c e n t r a t i o n s of t h e s u b s t r a t e s : L - g l u t a m a t e , 2 0 . 0 p M ; L - l e u c i n e , 21.7 ~ M ; L - g l u t a m i n e , 8 3 . 3 ~ M ; L - l a c t a t e , 1 3 3 . 0 ~uM; a c e t a t e , 7 8 . 0 p M ; a n d RbC1, 2 raM. T h e u p t a k e o f a c e t a t e a n d R b + w a s m e a s u r e d as s t e a d y - s t a t e levels ( n m o l / m g p r o t e i n ) i n s t e a d o f initial r a t e s , a f t e r 6 a n d 2 r a i n , r e s p e c t i v e l y . B l a n k s o b t a i n e d in t h e p r e s e n c e o f n i g e r i c i n ( 0 . 5 ~zM) w h i c h c o m p l e t e l y a b o l i s h e s t h e p H g r a d i e n t o v e r t h e m e m b r a n e [ 2 0 ] , w e r e subt r a c t e d f r o m t h e v a l u e s o b t a i n e d w i t h a c e t a t e . M o r e o v e r t h e s e v a l u e s r e p r e s e n t o n l y m i n i m u m o n e s , since t h e label is r a p i d l y l o s t f r o m t h e v e s i c l e s d u r i n g t h e w a s h o n t h e f i l t e r [ 2 0 ] . T o m e a s u r e t h e u p t a k e o f R b +, s o d i u m - l o a d e d vesicles ( 0 . 2 1 m g p r o t e i n p e r m l ) a n d s o d i u m p h o s p h a t e b u f f e r s w e r e a p p l i e d , a n d 150 arbitrary units of staphylococcin 1580 per ml (710 arbitrary units per mg membrane protein) were u s e d . T h e u p t a k e w a s e x p r e s s e d r e l a t i v e t o v a l u e s o b t a i n e d in t h e a b s e n c e o f v a l i n o m y c i n . Substrate
L-Glutamate L-Glutamate L-Glutamate L-Leucine L-Glut amine L-Lactate Acetate R b ÷ (in t h e p r e s e n c e o f v a l i n o m y c i n )
Electron donor system
ascorbate + PMS succinate + PMS NADH NADH NADH NAD H NADH NADH
Uptake (nmol/min per mg protein) --Staphylococcin 1580
+Stapbylococcin 1580
12.80 4.2.0 10.48 1.00 1.33 4.30 0.21 30.4
11.20 3.59 8.59 0.78 0.57 1.53 0.15 24.0
Inhibition (%)
12.6 14.5 10.5 22.0 57.2 64.5 28.5 21.0
392
not simply be traced down to differential effects of staphylococcin 1580 on the membrane pH gradient (uptake of acetate) and on the membrane potential (uptake of rubidium in the presence of valinomycin). This shows that staphylococcin 1580 has a differential effect on either the functioning of the various transport carriers or their coupling to the high-energy state of the membrane.
Effect of staphylococcin 1580 on anaerobic influx Fig. 3 shows that under anaerobic conditions, which strongly inhibited the active accumulation of L-glutamate and the oxidation of NADH [23], externally added L-glutamate does not completely equilibrate with the intramembranal pool in the presence of staphylococcin 1580. An alternative interpretation of the data might be that the label is rapidly lost from staphylococcintreated vesicles during the washing procedure. However, two observations favoured the first given conclusion: (i) Similar experiments were carried out without this washing and the a m o u n t of extravesicular water remaining on the filters was monitored with [3H]inulin, which is presumably excluded by the vesicles. The results were somewhat variable, but resemble those shown in Fig. 3. (ii) The rate of efflux of label during the washing procedure was similar for staphylococcin-treated and untreated vesicles (result not shown). The bacteriocin concentrations required to induce a distinct effect on the
UPTAKE [ nmoles / mg PROTEIN ] ...I-o-----o
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F i g . 3. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n t h e i n f l u x o f L - g l u t a m a t e i n m e m b r a n e v e s i c l e s u n d e r a n a e r o b i c conditions. Membrane vesicles (1.88 mg protein per ml) were incubated at 25°C in a final volume of 225 ~1 u n d e r a s t r e a m o f n i t r o g e n . A f t e r 2 m i n N A D H ( f i n a l c o n c e n t r a t i o n 5 mM) was added, and the mixt u r e w a s i n c u b a t e d a n a e r o b i c a l l y for 5 m i n m o r e . T h e n s t a p h y l o c o c c i n 1580 was added, followed 3 rain later b y t h e l a b e l l e d L - g l u t a m a t e . S a m p l e s ( 2 5 pl) w e r e r e m o v e d a t v a r i o u s t i m e i n t e r v a l s , w a s h e d w i t h 2 m l 0 . 1 M LiC1 a n d t h e r a d i o a c t i v i t y w a s d e t e r m i n e d . T h e f i n a l c o n c e n t r a t i o n o f L - g l u t a m a t e w a s 2 m M ( 4 . 9 C i / m o l ) . S y m b o l s : 0 ( o ) ; 8 U~); 3 4 (A); 8 5 (L1); 3 3 7 ( i ) arbitrary u n i t s o f s t a p h y l o c o c c i n 1 5 8 0 p e r m l .
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glutamine influx are much lower than those required to inhibit active glutamate uptake, which is particularly clear when the relatively high membrane concentrations used in the anaerobic experiments are taken into account. Effect o f staphylococcin 1580 on the L-glutamate carrier The effect of staphylococcin 1580 on the efflux of preaccumulated L-glutamate from membrane vesicles, was studied by diluting bacteriocin-treated and untreated vesicles 50-fold, w i t h o u t interfering with the energy supply (Fig. 4). The rate of glutamate efflux is distinctly higher in the staphylococcin-treated vesicles, and apparently the newly reached steady-state level is lower than in control vesicles. When the NADH concentration was simultaneously diluted 50-fold, and thus the NADH oxidation rate was lowered, comparable results were obtained. Fig. 5 shows the effect of staphylococcin 1580 on the exchange properties of the vesicles. Preaccumulated '4C-labelled L-glutamate exchanged slightly more rapidly with exogenously added unlabelled L-glutamate when the vesicles were treated first with rather high amounts of staphylococcin 1580.
% RESIDUAL GLUTAMATE 100
80
60
~0
20
i
I
L
I
I
i
0
I
2
3
4
5
TIME [rnin] F i g . 4. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n t h e L - g l u t a m a t e c a r r i e r s y s t e m u n d e r e f f l u x c o n d i t i o n s . M e m b r a n e vesicles ( 0 . 3 5 m g p r o t e i n p e r rnl) w e r e p r e l o a d e d f o r 8 r a i n w i t h 1 4 C - l a b e U e d L - g l u t a m a t e ( 2 0 . 0 ~ M ) in t h e p r e s e n c e o f 5 m M N A D H . F i n a l v o l u m e o f t h e i n c u b a t i o n m i x t u r e w a s 1 1 0 pl. S u b s e q u e n t l y , s t a p h y l o c o c c i n 1580 (250 arbitrary units per ml) or only b u f f e r were added and 2 rain later the incubat i o n m i x t u r e s w e r e r a p i d l y d i l u t e d 5 0 - f o l d w i t h p r e w a r m e d i n c u b a t i o n b u f f e r , c o n t a i n i n g e i t h e r 5 rnM or n o N A D H , a n d [ 3 H ] i n u h n ( 0 . 5 ~ C i / m l ) . S a m p l e s (1 m l ) w e r e t a k e n at v a r i o u s t i m e i n t e r v a l s a n d f i l t e r e d w i t h o u t f u r t h e r w a s h i n g . T h e a m o u n t o f e x t r a v e s i c u l a r w a t e r r e m a i n i n g on t h e f i l t e r w a s c a l c u l a t e d f r o m t h e 3 H r a d i o a c t i v i t y . T h e r e s u l t s are e x p r e s s e d as p e r c e n t a g e L - g l u t a m a t e r e m a i n i n g i n s t a p h y l o e o c c i n t r e a t e d or u n t r e a t e d v e s i c l e s a f t e r d i l u t i o n , w i t h r e g a r d t o t h e L - g l u t a m a t e c o n c e n t r a t i o n in v e s i c l e s t h a t w e r e n o t d i l u t e d . S y m b o l s : o, c o n t r o l s d i l u t e d in m e d i u m c o n t a i n i n g 5 m M N A D H ; ~, s t a p h y l o c o c c i n 1 5 8 0 t r e a t e d , d i l u t e d in m e d i u m c o n t a i n i n g 5 m M N A D H ; e, c o n t r o l s , d U u t e d in m e d i u m w i t h o u t N A D H ( r e s u l t i n g in a final c o n c e n t r a t i o n o f 0.1 m M ; A s t a p h y l o c o c c i n 1 5 8 0 t r e a t e d , d i l u t e d in m e d i u m w i t h o u t N A D H ( f i n a l c o n c e n t r a t i o n 0.1 m M ) .
394
% RESIDUAL GLUTAMATE I0C
80
60
~A /.0
20
I
I
I
0
1
2
I
I
3 /-. TIME [mini
I 5
F i g . 5. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n L - g l u t a m a t e e x c h a n g e . T h e c o n d i t i o n s w e r e t h e s a m e as in F i g . 4, e x c e p t t h a t t h e v e s i c l e s w e r e d i l u t e d 5 0 - f o l d in a b u f f e r m e d i u m c o n t a i n i n g 5 m M N A D H a n d 2 m M u n labelled L-glutamate. Symbols: e, vesicles not treated with staphylococcin 1580; A treated with 100 arbitrary units per ml; D treated with 250 arbitrary units per ml.
The results show that staphylococcin 1580 has a slight effect on the L-glutamate carrier function, either directly affecting the catalytic properties or uncoupling the carrier from the energy supply.
Effect o f staphylococcin 1580 on 3,3'-dipropylthiadicarbocyanine fluorescence Recently, Bhattacharryya et al. [ 12] used the fluorescent dye 3,3'-dipropylthiadicarboeyanine to m o n i t o r the membrane potential in membrane vesicles of A. vinelandii. The fluorescence quench can be directly correlated to the membrane potential (in mV) by calibration with the diffusion potential posed on potassium-loaded vesicles upon addition of valinomycin. Our results (not shown) obtained with membrane vesicles of B. subtilis were similar to those reported by Bhattacharryya et al. [12]. Fig. 6 shows that vesicles quenched the 3,3'-dipropylthiadicarbocyanine fluorescence upon addition of NADH (steady state 99 mV). Addition of staphylococcin 1580 instantaneously abolished the fluorescence quench to a final level with depends on the a m o u n t of bacteriocin applied; the resulting membrane potential was 79 and 40 mV after treatment with 500 and 2500 arbitrary units of staphylococcin 1580 per mg membrane protein, respectively. Staphylococcin 1580 abolishes also the membrane potential built up by the diffusion potential of K ÷ (Fig. 7). In the presence of the bacteriocin a lower optimum was reached and the quench was dissipated more rapidly. Almost no influence of staphylococcin 1580 was observed on the 3,3'-dipropylthiadicarbocyanine fluorescence with "resting" vesicles irrespective of sodium- or potassium-loaded vesicles were used in the presence of either
395 FLUORESCENCE [arbitrary units1
FLUORESCENCE]arbffrery units]
80
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t
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f
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gramD
I
I
I
I
I
1
2
3
z. TIME [rain]
5
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3
l. TIME [mini
Fig. 6. E f f e c t of s t a p h y l o c o c c i n 1 5 8 0 o n t h e r e s p i r a t i o n i n d u c e d q u e n c h i n g o f 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e (DiS-C3) f l u o r e s c e n c e . M e m b r a n e vesicles ( 2 0 p g p r o t e i n / m l ) w e r e i n c u b a t e d at 2 5 ° C in 50 m M p o t a s s i u m p h o s p h a t e ( p H 6.6), 10 m M MgSO 4 a n d 0.8 p M 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e . R e s p i r a t i o n was s t a r t e d b y t h e a d d i t i o n of 5 m M N A D H . W h e n t h e f l u o r e s c e n c e q u e n c h r e a c h e d a p l a t e a u , s t a p h y l o c o c c i n 1 5 8 0 in a final c o n c e n t r a t i o n of 2 5 0 0 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ( d o t t e d line), o r 500 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ( b r o k e n line), or only b u f f e r (solid line) w a s a d d e d . A t t h e e n d o f the e x p e r i m e n t g r a m i c i d i n D (1 # g / m l ) was a d d e d , w h i c h c o m p l e t e l y abolishes t h e m e m b r a n e potential. Fig. 7. E f f e c t of s t a p h y l o c o c c i n 1 5 8 0 o n t h e 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e (DiS-C 2 - f l u o r e s c e n c e q u e n c h i n g , i n d u c e d b y a p o t a s s i u m d i f f u s i o n p o t e n t i a l . P o t a s s i u m - l o a d e d m e m b r a n e vesicles ( 2 0 pg protein p e r m l ) w e r e i n c u b a t e d f o r 2 m i n at 25°C in a b u f f e r c o n t a i n i n g 49 m M s o d i u m p h o s p h a t e a n d 1 m M p o t a s s i u m p h o s p h a t e ( p H 6 . 6 ) , 10 m M M g S O 4 , a n d s t a p h y l o c o c c i n 1 5 8 0 in a final c o n c e n t r a t i o n o f 2 5 0 0 ( d o t t e d line) a n d 500 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ( b r o k e n line) o r b u f f e r i n s t e a d o f t h e bact e r i o c i n (solid line). T h e n 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e (0.8 #M) was a d d e d , f o l l o w e d a b o u t 1 m i n l a t e r b y v a l i n o m y c i n (0.5/~M). A t t h e e n d o f the e x p e r i m e n t g r a m i e i d i n D (1 p g / m l ) was a d d e d .
sodium or potassium phosphate buffer. T he refore staphylococcin 1580 cann o t be regarded as a specific c o n d u c t o r of either K ÷ or Na ÷.
Discussion Many observations have poi nt e d to the cytoplasmic m e m b r a n e as the target for the action o f staphylococcin 1580 [2,3,5]. T h e r e f o r e a n u m b e r of effects o f the bacteriocin on isolated m e m b r a n e vesicles of B. subtilis were studied and c o m p a r e d to the effects on whole cells. Konings [11,12] has shown t hat the m e m b r a n e o f B. subtilis W23 vesicles, pr e pa red by the m e t h o d used in this study, are topologically identical to the membranes of intact cells. Moreover, the specific transport rates of these vesicles approxi m at e those of intact cells when expressed as mol t r a ns por t e d per weight unit of m e m b r a n e protein. However, some structural and functional differences between cells and m em brane vesicles affect their susceptibility to staphylococcin 1580. The access of the bacteriocin to the m e m b r a n e could be impeded in whole cells due t o the pres-
396 ence of cell wall aspecific binding sites for the bacteriocin [22]. Moreover, the complex balance between intra- and extracellular solutes in whole cells may result in differences in the sensitivity to the bacteriocin on comparing whole cells with m e m b r a n e vesicles. This may be illustrated by the suggestion of Brewer [24] th at after colicin K t r e a t m e n t the membrane potential of E. coli cells depends on an electrochemical potential of an anion. In accordance, anions, such as pyruvate and p h o s p h o r y l a t e d intermediates of glycolysis, are lost after colicin E1 or K t r e a t m e n t [25]. The results obtained in this investigation allow a classification of the effects of staphylococcin 1580 into at least three groups (Table II). The anaerobic influx o f L-glutamate in m em br ane vesicles is most sensitive to the bacteriocin. At present, the way in which staphylococcin 1580 affects this influx is not clear. The kinetics of solute permeation has been extensively studied with m e mb r an e vesicles of E. coli [26,27] and S. aureus [28]. In S. aureus vesicles serine efflux, and influx under energized conditions, exhibited saturation kinetics. However, anaerobic influx appeared to be a non-saturable process, and it was co n clu d ed that the transport carriers catalyze facilitated diffusion in the direction of efflux only [28]. Extending this conclusion to L-glutamate influx in B. subtilis vesicles would mean that staphylococcin 1580 specifically blocks this passive diffusion. Such an effect could be explained by a change in membrane charge or h y d r o p h o b i c i t y of the diffusion barrier. A second group of effects are exerted by staphylococcin 1580 when applied to cells or m e m b r a n e vesicles in concentrations of about 250 and 4 0 0 - - ! 6 0 0 arbitrary units per mg of m e m b r a n e protein, respectively. These effects can be explained by a dissipation of the p r o t o n motive force since A~ (rubidium uptake in the presence of valinomycin; 3,3'-dipropylthiadicarbocyanine fluorescence) and ApH (acetate uptake) of m em br ane vesicles are abolished by these bacteriocin concentrations. The observed differences between the inhibitory effects on individual t r ans por t systems probably result from (i) a different sensing of the transport carriers to the energy supply, and (ii) a difference in direct susceptibility of the carrier to the bacteriocin, since it was shown to affect the L-glutamate carrier. Whole cells were shown to be a b o u t five times m ore sensitive to staphylococcin 1580 than vesicles, when the effect of the bacterio-
T A B L E II CLASSIFICATION OF EFFECTS AND MEMBRANE VESICLES
INDUCED BY STAPHYLOCOCCIN
1 5 8 0 IN B. S U B T 1 L I S
CELLS
System
Process
Amount of bacteriocin yielding a half-maximal effect (arbitrary units/ mg membrane protein)
Vesicles Vesicles
A n a e r o b i c L - g l u t a m a t e i n f l u x , s t e a d y - s t a t e level A c t i v e u p t a k e o f s o l u t e s c o u p l e d to t h e h i g h - e n e r g y s t a t e o f the membrane * ; membrane potential (fluorescence); L-glutamate efflux L - G l u t a m a t e u p t a k e ; killing a-Methylglucoside uptake
20---60 400---1600
Cells Cells
* S o l u t e s listed in T a b l e I.
250 9400
397
cin on the uptake of L-glutamate was measured. This may reflect the complex composition of the membrane potential in whole cells. Inhibition of ~-methylglucoside uptake in B. subtilis cells required the application of distinctly higher concentrations of staphylococcin 1580. This seems to contradict the previously reported inhibition of o-nitrophenyl-~-galactoside hydrolysis in S. aureus, which is also mediated by the p h o s p h o e n o l p y r u v a t e phosphotransferase system [29], by moderate staphylococcin concentrations. However, it may reflect a different regulation of uptake systems in both organisms, rather than the induction of gross permeability changes in S. aureus. Moreover, the present results are in accordance with the observations made with colicins E1 and K [18] and Ia [19]. References 1 L u r i a , S.E. ( 1 9 7 3 ) In B a c t e r i a l M e m b r a n e s a n d Walls (Leive, L., ed.), p p . 2 9 3 - - 3 3 0 , M a r c e l D e k k e r , Inc., New York 2 J e t t e n , A.M. a n d V o g e l s , G . D . ( 1 9 7 2 ) A n t i m i c r o b . A g e n t s C h e m o t h e r . 2, 4 5 6 - - 4 6 3 3 J e t t e n , A.M. a n d V o g e l s , G . D . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 1 , 4 8 3 - - 4 9 5 4 W e e r k a m p , A., G e e r t s , W. a n d V o g e l s , G . D . ( 1 9 7 7 ) A n t i m i e r o b . A g e n t s C h e m o t h e r . 1 2 , 3 1 4 - - 3 2 1 5 W e e r k a m p , A. a n d V o g e l s , G . D . ( 1 9 7 8 ) A n t i m i c r o b . A g e n t s C h e m o t h e r . , in t h e p r e s s 6 B h a t t a c h a r r y y a , P., W e n d t , L., W h i t n e y , E. a n d Silver, S. ( 1 9 7 0 ) S c i e n c e 1 6 8 , 9 9 8 - - 1 0 0 0 7 B i s s c h o p , A., D o d d e m a , H. a n d K o n i n g s , W.N. ( 1 9 7 5 ) J. B a c t e r i o l . 1 2 4 , 6 1 3 - - 6 2 2 8 K o n i n g s , W . N . , B i s s c h o p , A. a n d D a a t s e l a a r , M.C.C. ( 1 9 7 2 ) F E B S L e t t . 2 4 , 2 6 0 - - 2 6 4 9 M a t i n , A. a n d K o n i n g s , W.N. ( 1 9 7 3 ) E u r . J. B i o c h e m . 3 4 , 5 8 - - 6 7 1 0 F o r t n a g e l , P. a n d F r e e s e , E. ( 1 9 6 8 ) J. B a c t e r i o l . 9 5 , 1 4 3 1 - - 1 4 3 8 11 K o n i n g s , W . N . , B i s s c h o p , A . , V e e n h u i s , M. a n d V e r m e u l e n , C . A . ( 1 9 7 3 ) J. B a c t e r i o l . 1 1 6 , 1 4 5 6 - 1465 12 B h a t t a e h a r r y y a , P., S h a p i r o , S.A. a n d B a r n e s , J r . , E.M. ( 1 9 7 7 ) J. B a c t e r i o l . 1 2 9 , 7 5 6 - - 7 6 2 1 3 W a g g o n e r , A. ( 1 9 7 6 ) J. M e m b r a n e Biol. 2 7 , 3 1 7 - - 3 3 4 1 4 J e t t e n , A . M . , V o g e l s , G . D . a n d d e W i n d t , F. ( 1 9 7 2 ) J. B a c t e r i o l . 1 1 2 , 2 3 5 - - 2 4 2 1 5 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 1 6 K a b a c k , H . R . ( 1 9 7 1 ) In M e t h o d s in E n z y m o l o g y ( J a c o b y , W.B., ed.), V o l . 2 2 , p p . 9 9 - - 1 2 0 , A c a d e m i c Press, N e w Y o r k 17 D e l o b b e , A., H a g u e n a u e r , R. a n d R a p o p o r t , G. ( 1 9 7 1 ) B i o c h i m i e 5 3 , 1 0 1 5 - - 1 0 2 1 1 8 J e t t e n , A.M. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 4 0 , 4 0 3 - - 4 1 1 1 9 G i l c h r i s t , M. a n d K o n i s k y , J. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 2 4 5 7 - - 2 4 6 2 2 0 R a m o s , S., S e h u l d i n e r , S. a n d K a b a c k , H . R . ( 1 9 7 6 ) P r o c . N a t l . A c a d . Sci. U.S. 7 3 , 1 8 9 2 - - 2 8 9 6 21 K o n i n g s , W.N. ( 1 9 7 5 ) A r c h . B i o c h e m . B i o p h y s . 1 6 7 , 5 7 0 - 5 8 0 2 2 J e t t e n , A.M. a n d Vogels, G . D . ( 1 9 7 2 ) J. B a c t e r i o l . 1 1 2 , 2 4 3 - - 2 5 0 2 3 B i s s e h o p , A., B o o n s t r a , J., S i p s , H . J . a n d K o n i n g s , W.N. ( 1 9 7 5 ) F E B S L e t t . 6 0 , 1 1 - - 1 6 24 Brewer, G.J. (1976) Biochemistry 15, 1387--1392 2 5 Fields, K . L . a n d L u r i a , S.E. ( 1 9 6 9 ) J. B a c t e r i o l . 9 7 , 6 4 - - 7 7 2 6 K a b a e k , H . R . a n d B a r n e s , J r . , E.M. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 5 5 2 3 - - 5 5 3 1 27 L o m b a r d i , F . J . a n d K a b a c k , H . R . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 8 4 4 - - 7 8 5 7 28 S h o r t , S . A . a n d K a b a c k , H . R . ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 4 2 7 5 - 4 2 8 1 29 K e n n e d y , E.P. a n d S c a r b o r o u g h , G . A . ( 1 9 6 7 ) P r o c . N a t l . A c a d . Sci. U.S. 5 8 , 2 2 5 - - 2 2 8
Biochimica et Biophysica 4cta, 539 (1978) 386--397 © Elsevier/North-Holland Biomedical Press
BBA 28453 E F F E C T S OF STAPHYLOCOCCIN 1580 ON CELLS AND MEMBRANE VESICLES OF B A C I L L U S S U B T I L I S W23
A. WEERKAMP * and G.D. VOGELS Department of Microbiology, Faculty of Science, University of Ni]megen, Ni]megen (The Netherlands)
(Received August 16th, 1977)
Summary 1. Uptake o f L-glutamic acid is inhibited, and preaccumulated L-glutamic acid is released from Bacillus subtilis cells treated with staphylococcin 1580. Uptake of a-methylglycoside is enhanced at low bacteriocin concentrations and inhibited by excess bacteriocin. 2. Inhibition of amino acid uptake into m e m b r a n e vesicles is somewhat less sensitive to staphylococcin 1580 than uptake into whole cells under similar conditions, when the bacteriocin c o n c e n t r a t i o n is expressed per weight unit of m e m br an e protein. Inhibition of uptake into vesicles is i n d e p e n d e n t of the electron d o n o r system used, but varies with different substrates. 3. Influx of L-glutamic acid into vesicles under anaerobic conditions is severely hampered by staphylococcin 1580. The L-glutamic acid carrier functions are slightly affected only. 4. Scaphylococcin 1580 abolished the m e m b r a n e potential induced by respiration or by a potassium diffusion potential in the presence of valinomycin, as measured with the fluorescent dye 3,3'-dipropylthiadicarbocyanine. 5. The effects of staphylococcin 1580 on cells and m e m b r a n e vesicles allowed the classification into three groups with different sensitivity to the staphylococcin concentration.
Introduction Bacteriocins are protein antibiotics, which are bactericidal to bacteria related to the producing strain. One class of bacteriocins is assumed to exert its action directly on the cytoplasmic membrane. Among them, especially colicins E1 and
* To whom correspondence should be directed at the present address: Department University of British Columbia, Vancouver B.C. V6T 1W5, Canada.
of Microbiology,
387 K [1] have been studied extensively. Also staphylococcin 1580, a bacteriocin p r o d u c e d by Staphylococcus epidermidis, has been studied to considerable depth [2--5]. Like colicins E1 and K, staphylococcin 1580 rapidly induces an inhibition of macromolecular syntheses and active transport, depletion of cellular ATP and leak of preaccumulated Rb ÷ from the cells [2]. However, the permeability to p r o t ons is n o t affected [3,5]. Sensitivity of cells to staphylococcin 1580 is d e p e n d e n t on the physiological state of the cells, and is influenced by conditions such as the t em perat ure during growth and incubation, energy charge of the cells and the kind of carbon source applied in the medium [5]. It was suggested that the primary effect of staphylococcin 1580 is the uncoupling of the m em brane potential and electron transport [5]. In spite of the advantages m e m b r a n e vesicles offer to study energy coupling and transport p h e n o m e n a , very few reports considered the effects of bacteriocins on vesicles [3,6]. J e t t e n and Vogels [3] showed that transport of amino acids in memb r an e vesicles was inhibited by staphylococcin 1580. This study aims to compare the action of staphylococcin 1580 on whole cells and membrane vesicles, and to e xt e nd the previous experiments as to the m ode of action o f the bacteriocin. In the present study Bacillus subtilis W23, an organism equally sensitive to staphylococcin 1580 as the previously used Staphylococcus aureus [3] was used as the indicator strain. Uptake of various substrates into vesicles prepared from B. subtilis W23 has been extensively studied by Konings et al. [7--9]. T h e y showed that these vesicles are essentially right side out and t hat transport rates of vesicles appr oxi m a t e those of intact cells. Materials and Methods
Bacterial strain, medium and growth conditions. B. subtilis W23 was grown in nutrient-sporulation medium [10] at 37°C with vigorous aeration. The medium was inoculated with an overnight grown culture to obtain an absorbance at 600 nm (A600nm) of 0.05, and harvested in mid-exponential phase (A600n m 1.2). The cells were washed once with 0.1 M potassium phosphate (pH 7.3), and centrifuged 10 min at 12 000 × g. Preparation of membrane vesicles. Membrane vesicles were prepared by direct lysis in h y p o t o n i c medium, as described by Konings and coworkers [7,11], with the e x c e p t i o n t hat the cells were harvested in the mid-exponential rather tt, n in the early stationary phase. Sodium-loaded vesicles were prepared by an identical procedure, e x c e p t that sodium buffers replaced potassium buffers t h r o u g h o u t the procedure. Vesicles were rapidly frozen in thinwalled plastic tubes by the use of liquid nitrogen and stored a t - - 8 0 ° C . Immediately before use vesicles were rapidly thawed at 46°C in a water bath; only vesicles th at were frozen once were used. Transport experiments. Incubation mixtures contained, unless otherwise indicated, final concentrations of either 50 mM potassium or sodium phosphate (pH 6.6), 10 mM MgSO4 and 0.2--0.5 mg of m e m b r a n e protein per ml in a total volume o f 50--250 gl. Mixtures were incubated at 25°C with a stream of water-
388 saturated oxygen blowing over the surface. After 2 min preincubation the electron d o n o r was added, followed I min later by the labelled substrate. Initial uptake rates were measured after 30 s by terminating the reaction by the addition o f 2 ml 0.1 M LiC1, as described earlier [3]. The time course of uptake was measured by removing samples from the incubation m i xt ure at various time intervals with an O x f o r d sampler (Oxford Lab. Intern. Corp.). Samples were spotted on a p r e w e t t e d 0.45 gm pore size m e m b r a n e filter (Millipore Corp.) and washed with 3 ml 0.1 M LiC1, at r o o m temperature. Uptake experiments in whole cells (about 1.2 mg dry weight per ml) were p e rf o r med as described for m e m b r a n e vesicles. Fluorescence measurements. Fluorescence of 3,3'-dipropylthiadicarbocyanine was used to m o n i t o r the m em br ane potential [12,13] and was carried out as described previously [ 5]. The incubation mixtures (2.5 ml final volume) contained 50 mM of either sodium or potassium phosphate (pH 6.6), 10 mM MgSO4 and 20 pg m e m b r a n e protein per ml. 3,3'-Dipropylthiadicarbocyanine was added to a c o n c e n t r a t i o n of 0.8 ~M and fluorescence was excited at 620 nm and measured at 662 nm, in an Aminco Bowman spectrofluorimeter. Measurements were carried out at 25°C. The percent fluorescence quenched was calibrated with a potassium diffusion potential to estimate the respirationinduced memb r an e potential. Potassium-loaded vesicles were diluted into phosphate buffers containing different p r o p o r t i o n s of sodium and potassium phosphate. The fluorescence quench occurring u pon addition of valinomycin was recorded and p lo tt e d against the diffusion potential, calculated from the Nernst equation (A~ = 59 log K~n/Kout). Our results were similar to those obtained by B h a t t a c h a r r y y a et al. [12] who used Azotobacter vinelandii vesicles. Preparation and assay o f staphylococcin 1580. Product i on and purification o f the bacteriocin were described previously [4]. Briefly, the bacteriocin was isolated from supernatants of S. epidermidis 1580 cultures in medium A with 1% sodium pyruvate, and purified by a m m o n i u m sulfate precipitation and chrom a t o g r a p h y on CM-Sephadex C25. The final preparation was hom ogeneous on polyacrylamide gel electrophoresis and isoelectrofocussing, and usually contained a b o u t 25 000 arbitrary units per mg protein. Activity was measured as described previously [14] and expressed as arbitrary units per ml. Determination o f viability. Cells were incubated at 25°C as described for the uptake experiments. After 2 min of preincubation, staphylococcin 1580 was added and after a not her 4-min samples were removed, rapidly diluted 1000fold in p e p t o n e water (Oxoid Ltd, L o n d o n ) and plated in suitable dilutions on n utr ien t agar (Oxoid) containing 0.2 M NaC1. The results were expressed as percent survivors relative to cell suspensions n o t treated with staphylococcin 1580. Protein determination. Protein was measured according to the m e t h o d of L o w ry et al. [15]. The m em br ane protein c o n t e n t of whole cells was assumed to be 15% of the total protein c o n t e n t [16]. Chemicals. 3,3'-Dipropylthiadicarbocyanine was a gift from A. Waggoner, Amherst College, Amherst, Mass. Nigericin was donat ed by Dr. W.E. Scott, H o f f m a n La Roche, Nutley, N.J. Valinomycin was obtained from Calbiochem, Lucerne, Switzerland; gramicidin D was from Boehringer, Mannheim, G.F.R.,
389 and 5-N-methylphenazonium methosulfate from B.D.H. Chemicals Ltd., Poole, U.K. Radiochemicals, obtained from Radiochemical Centre, Amersham, U.K., were L-[UJ4C]glutamic acid (270 Ci/mol), L-[UJ4C]glutamine (40 Ci/mol), L-[U-14C]leucine (330 Ci/mol), L-[UJ4C]lactate (sodium salt) (50 Ci/mol), [1J4C]acetate (sodium salt) (58 Ci/mol), [3H]inulin (695 Ci/mol), S6RbC1 (2-10 Ci/g), and methyl (a-D-[UJ4C] gluco)pyranoside (3 Ci/mol). Results
Effect o f staphylococcin 1580 on active transport in whole cells and membrane vesicles o f B. su btilis It was previously shown that staphylococcin 1580 blocks active uptake of amino acids in cells [2] and membrane vesicles [3] of S. sureus, and causes the efflux of preaccumulated solute. Fig. 1 shows that uptake of L-glutamate in cells of B. subtilis is inhibited and that the preaccumulated amino acid is rapidly released under the influence of the bacteriocin. However, similar concentrations of staphylococcin 1580 have much less severe effect on the uptake of ~-methylglucoside. Relatively low concentrations of staphylococcin 1580 even enhance uptake significantly, up to a maximal stimulation of 40% under the conditions tested. In B. subtilis ~-methylglucoside is transported by the phosphoenolpyruvate:glucose phosphotransferase system, and retained within the cell both as ~-methylglucoside phosphate and, after dephosphorylation, as ~-methylglucoside, which are not further metabolized under the experimental
UPTAKE[nmoles/mgMEMBRANEPROTEIN] 50f
500
L-GIu
LO
/" / "
30
~MG
LO0 300
/
20
200
10
100 /
/
,
f 2
4
6
0
I
2
I
L
l. 6 TIME[min]
F i g . 1. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n t h e u p t a k e o f L - g l u t a m a t e ( L - G l u ) a n d ~ - m e t h y l g l u c o s i d e (c~MG) b y cells o f B. s u b t i l i s W 2 3 . Cells w e r e g r o w n i n t o m i d - e x p o n e n t i a l p h a s e , w a s h e d a n d r e s u s p e n d e d in 50 m M p o t a s s i u m p h o s p h a t e ( p H 6 . 6 ) , a n d 10 m M M g S O 4, at a final c o n c e n t r a t i o n o f 1 . 2 m g d r y w e i g h t p e r m l . U P t a k e e x p e r i m e n t s w e r e c a r r i e d o u t a t 2 5 ° C as d e s c r i b e d , b u t an e x o g e n o u s e n e r g y s o u r c e w a s o m i t t e d . S t a p h y l o c o c c i n 1 5 8 0 w a s a d d e d 2.5 r a i n ( i n d i c a t e d b y t h e a r r o w ) a f t e r t h e a d d i t i o n o f t h e label in a final c o n c e n t r a t i o n o f 10 ( o ) a n d 3 5 0 a r b i t r a r y u n i t s p e r m l (A), r e s p e c t i v e l y . C o n t r o l s r e c e i v e d n o s t a p h y l o c o c c i n ( e ) . T h e u p t a k e of L - g l u t a m a t e ( 1 1 . 7 p M ) a n d ~ - m e t h y l g l u c o s i d e ( 1 . 5 r a M ) w a s p l o t t e d against the time.
390
SOLUTE UPTAKE [% of control] - VIABILITY
100 -
6O
2O
1
2
3
L,
LOG AU./rag MEMBRANE PROTEIN Fig. 2. C o r r e l a t i o n b e t w e e n t h e c o n c e n t r a t i o n o f s t a p h y l o c o c c i n 1 5 8 0 a n d i t s e f f e c t o n t h e v i a b i l i t y of c e l l s , o n L - g l u t a m a t e u p t a k e in cells a n d m e m b r a n e vesicles a n d o n ( ~ - m e t h y l g l u c o s i d e u p t a k e in cells. Upt a k e r a t e s o f L - g l u t a m a t e ( 1 1 . 7 p M ) a n d e - m e t h y l g l u c o s d e ( 1 . 5 r a M ) w e r e m e a s u r e d a f t e r 30 s, a n d a m o u n t e d t o 1 2 . 2 a n d 1 1 5 . 0 n m o l / m i n p e r m g m e m b r a n e p r o t e i n , r e s p e c t i v e l y , w h e r e a s 9 . 4 n m o l L-glut a m a t e w e r e t a k e n u p p e r r a i n a n d p e r m g m e m b r a n e p r o t e i n in t e s t s w i t h vesicles. S t a p h y l o e o c c i n 1 5 8 0 , d i s s o l v e d in b u f f e r , w a s a d d e d 1 r a i n p r i o r t o t h e a d d i t i o n o f t h e l a b e l l e d c o m p o u n d ; c o n t r o l s r e c e i v e d o n l y t h e b u f f e r . U p t a k e in m e m b r a n e s vesicles w a s s t i m u l a t e d b y s o d i u m a s c o r b a t e ( 2 0 r a M ) a n d 5-Nm e t h y l p h e n a z o n i u m m e t h o s u l f a t c ( P M S ) (0.1 m M ) ; in e x p e r i m e n t s w i t h w h o l e cells no e x o g e n o u s e n e r g y s o u r c e w a s a d d e d . T h e v i a b i l i t y w a s m e a s u r e d a f t e r t r e a t m e n t o f t h e cells w i t h s t a p h y l o c o c c i n f o r 4 rain. R e s u l t s w e r e e x p r e s s e d as t h e p e r c e n t a g e o f c o n t r o l e x p e r i m e n t s w i t h o u t s t a p h y l o c o c c i n 1 5 8 0 , a n d p l o t t e d as a f u n c t i o n o f t h e s t a p h y l o c o c c i n c o n c e n t r a t i o n . O p e n s y m b o l s : ©, L - g l u t a m a t e u p t a k e , w h o l e cells; A ~ - m e t h y l g l u c o s i d e u p t a k e ; D v i a b i l i t y . C l o s e d s y m b o l s : • L - g l u t a m a t e u p t a k e , m e m b r a n e vesicles.
conditions [17]. The r e t e nt i on of a-methylglucoside within the cells indicates that the integrity of the cell m e m b r a n e is conserved when staphylococcin was applied in concentrations t hat effectively inhibited glutamate uptake. The uptake o f a-methylglucoside was inhibited when higher concentrations o f the bacteriocin were used (Fig. 2). This effect is presumably due to an increase of the permeability of the membrane. Similar results have been reported for Escherichia coli cells treated with colicins E1 and K [18] or Ia [19], except that several-fold stimulations were f o u n d with cells grown on glycerol. The killing o f the cells correlates with the inhibition of L-glutamate uptake (Fig. 2) if the bacteriocin c o n c e n t r a t i o n is expressed as arbitrary units per mg m e m b r a n e protein. This way of plotting was chosen in order to compare the effect o f staphylococcin 1580 on bot h cells and m e m b r a n e vesicles and is based on the assumption t hat the m e m b r a n e is the direct target for the action of staphylococcin 1580. If expressed in this way transport rates for L-gluta-
391
mate were a b o u t similar in whole cells and m e m b r a n e vesicles [11,21], b u t a five times higher staphylococcin 1580 c o n c e n t r a t i o n is required to inhibit t r a n s p o r t in m e m b r a n e vesicles to the same e x t e n t as in whole cells. This difference presumably is n o t the result o f a difference in the a m o u n t of staphylococcin 1580 adsorbed by either cells or vesicles, since whole cells can adsorb more bacteriocin than vesicles can (under the experimental conditions a b o u t 750 and 250 arbitrary units per mg m e m b r a n e protein, respectively). It was previously shown t ha t staphylococcin 1580 has a strong t e n d e n c y to stick aspecifically to both cell walls and membranes of sensitive as well as resistant bacteria [22]. It should be noted, however, t hat the ratio between specific and unspecific adsorption is u n k n o w n . Table I shows the ef f ect of staphylococcin 1580 on the uptake of various c o m p o u n d s by m e m b r a n e vesicles, and the influence of various electron d o n o r systems. The initial upt a ke of L-glutamate was inhibited to the same ext ent , whenever NADH, s ucci nat e/ 5- N - m et hyl phenazoni um methosulfate, or ascorb a t e / 5 - N - m e t h y l p h e n a z o n i u m m e t hos ul f a t e were used as electron donors. This result is conceivable, since staphylococcin 1580 does n o t affect the electron transport, or m e m b r a n e - b o u n d dehydrogenases [3]. The differences observed between the transport inhibition of various substrates by the bacteriocin can-
TABLE I E F F E C T O F S T A P H Y L O C O C C I N 1580 O N T H E U P T A K E O F V A R I O U S S U B S T R A T E S BY MEMB R A N K V E S I C L E S O F B. S U B T I L I S W 2 3 , E N E R G I Z E D B Y V A R I O U S E L E C T R O N D O N O R S P o t a s s i u m - l o a d e d v e s i c l e s ( 0 . 3 7 m g p r o t e i n p e r m l ) w e r e i n c u b a t e d at 2 5 ° C w i t h 50 m M p o t a s s i u m p h o s p h a t e ( p H 6 . 6 ) , a n d 10 m M M g S O 4 in a final v o l u m e o f 55 pl. 2 5 0 a r b i t r a x y u n i t s o f s t a p h y l o c o c c i n 1 5 8 0 ( 6 7 5 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ) w e r e a d d e d i m m e d i a t e l y p r i o r t o t h e a d d i t i o n o f t h e electron donors. A f t e r 1 m i n of i n c u b a t i o n the u p t a k e was started by the addition of the labelled c o m p o u n d s . T h e u p t a k e w a s t e r m i n a t e d 30 s l a t e r a n d t h e r a d i o a c t i v i t y w a s m e a s u r e d . T h e e l e c t r o n d o n o r s u s e d w e r e : N A D H ( 5 raM); s o d i u m a s c o r b a t e ( 2 0 r a M ) a n d 5 - N - m e t h y l p h c n y l a z o n i u m m e t h o s u l f a t e ( P M S ) (0.1 m M ) ; s o d i u m succinate (10 raM) and 5 o N - m e t h y l p h e n a z o n i u m m e t h o s u l f a t e (0.2 raM). Final c o n c e n t r a t i o n s of t h e s u b s t r a t e s : L - g l u t a m a t e , 2 0 . 0 p M ; L - l e u c i n e , 21.7 ~ M ; L - g l u t a m i n e , 8 3 . 3 ~ M ; L - l a c t a t e , 1 3 3 . 0 ~uM; a c e t a t e , 7 8 . 0 p M ; a n d RbC1, 2 raM. T h e u p t a k e o f a c e t a t e a n d R b + w a s m e a s u r e d as s t e a d y - s t a t e levels ( n m o l / m g p r o t e i n ) i n s t e a d o f initial r a t e s , a f t e r 6 a n d 2 r a i n , r e s p e c t i v e l y . B l a n k s o b t a i n e d in t h e p r e s e n c e o f n i g e r i c i n ( 0 . 5 ~zM) w h i c h c o m p l e t e l y a b o l i s h e s t h e p H g r a d i e n t o v e r t h e m e m b r a n e [ 2 0 ] , w e r e subt r a c t e d f r o m t h e v a l u e s o b t a i n e d w i t h a c e t a t e . M o r e o v e r t h e s e v a l u e s r e p r e s e n t o n l y m i n i m u m o n e s , since t h e label is r a p i d l y l o s t f r o m t h e v e s i c l e s d u r i n g t h e w a s h o n t h e f i l t e r [ 2 0 ] . T o m e a s u r e t h e u p t a k e o f R b +, s o d i u m - l o a d e d vesicles ( 0 . 2 1 m g p r o t e i n p e r m l ) a n d s o d i u m p h o s p h a t e b u f f e r s w e r e a p p l i e d , a n d 150 arbitrary units of staphylococcin 1580 per ml (710 arbitrary units per mg membrane protein) were u s e d . T h e u p t a k e w a s e x p r e s s e d r e l a t i v e t o v a l u e s o b t a i n e d in t h e a b s e n c e o f v a l i n o m y c i n . Substrate
L-Glutamate L-Glutamate L-Glutamate L-Leucine L-Glut amine L-Lactate Acetate R b ÷ (in t h e p r e s e n c e o f v a l i n o m y c i n )
Electron donor system
ascorbate + PMS succinate + PMS NADH NADH NADH NAD H NADH NADH
Uptake (nmol/min per mg protein) --Staphylococcin 1580
+Stapbylococcin 1580
12.80 4.2.0 10.48 1.00 1.33 4.30 0.21 30.4
11.20 3.59 8.59 0.78 0.57 1.53 0.15 24.0
Inhibition (%)
12.6 14.5 10.5 22.0 57.2 64.5 28.5 21.0
392
not simply be traced down to differential effects of staphylococcin 1580 on the membrane pH gradient (uptake of acetate) and on the membrane potential (uptake of rubidium in the presence of valinomycin). This shows that staphylococcin 1580 has a differential effect on either the functioning of the various transport carriers or their coupling to the high-energy state of the membrane.
Effect of staphylococcin 1580 on anaerobic influx Fig. 3 shows that under anaerobic conditions, which strongly inhibited the active accumulation of L-glutamate and the oxidation of NADH [23], externally added L-glutamate does not completely equilibrate with the intramembranal pool in the presence of staphylococcin 1580. An alternative interpretation of the data might be that the label is rapidly lost from staphylococcintreated vesicles during the washing procedure. However, two observations favoured the first given conclusion: (i) Similar experiments were carried out without this washing and the a m o u n t of extravesicular water remaining on the filters was monitored with [3H]inulin, which is presumably excluded by the vesicles. The results were somewhat variable, but resemble those shown in Fig. 3. (ii) The rate of efflux of label during the washing procedure was similar for staphylococcin-treated and untreated vesicles (result not shown). The bacteriocin concentrations required to induce a distinct effect on the
UPTAKE [ nmoles / mg PROTEIN ] ...I-o-----o
3
0
A
~
8
12 TINE [min]
F i g . 3. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n t h e i n f l u x o f L - g l u t a m a t e i n m e m b r a n e v e s i c l e s u n d e r a n a e r o b i c conditions. Membrane vesicles (1.88 mg protein per ml) were incubated at 25°C in a final volume of 225 ~1 u n d e r a s t r e a m o f n i t r o g e n . A f t e r 2 m i n N A D H ( f i n a l c o n c e n t r a t i o n 5 mM) was added, and the mixt u r e w a s i n c u b a t e d a n a e r o b i c a l l y for 5 m i n m o r e . T h e n s t a p h y l o c o c c i n 1580 was added, followed 3 rain later b y t h e l a b e l l e d L - g l u t a m a t e . S a m p l e s ( 2 5 pl) w e r e r e m o v e d a t v a r i o u s t i m e i n t e r v a l s , w a s h e d w i t h 2 m l 0 . 1 M LiC1 a n d t h e r a d i o a c t i v i t y w a s d e t e r m i n e d . T h e f i n a l c o n c e n t r a t i o n o f L - g l u t a m a t e w a s 2 m M ( 4 . 9 C i / m o l ) . S y m b o l s : 0 ( o ) ; 8 U~); 3 4 (A); 8 5 (L1); 3 3 7 ( i ) arbitrary u n i t s o f s t a p h y l o c o c c i n 1 5 8 0 p e r m l .
393
glutamine influx are much lower than those required to inhibit active glutamate uptake, which is particularly clear when the relatively high membrane concentrations used in the anaerobic experiments are taken into account. Effect o f staphylococcin 1580 on the L-glutamate carrier The effect of staphylococcin 1580 on the efflux of preaccumulated L-glutamate from membrane vesicles, was studied by diluting bacteriocin-treated and untreated vesicles 50-fold, w i t h o u t interfering with the energy supply (Fig. 4). The rate of glutamate efflux is distinctly higher in the staphylococcin-treated vesicles, and apparently the newly reached steady-state level is lower than in control vesicles. When the NADH concentration was simultaneously diluted 50-fold, and thus the NADH oxidation rate was lowered, comparable results were obtained. Fig. 5 shows the effect of staphylococcin 1580 on the exchange properties of the vesicles. Preaccumulated '4C-labelled L-glutamate exchanged slightly more rapidly with exogenously added unlabelled L-glutamate when the vesicles were treated first with rather high amounts of staphylococcin 1580.
% RESIDUAL GLUTAMATE 100
80
60
~0
20
i
I
L
I
I
i
0
I
2
3
4
5
TIME [rnin] F i g . 4. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n t h e L - g l u t a m a t e c a r r i e r s y s t e m u n d e r e f f l u x c o n d i t i o n s . M e m b r a n e vesicles ( 0 . 3 5 m g p r o t e i n p e r rnl) w e r e p r e l o a d e d f o r 8 r a i n w i t h 1 4 C - l a b e U e d L - g l u t a m a t e ( 2 0 . 0 ~ M ) in t h e p r e s e n c e o f 5 m M N A D H . F i n a l v o l u m e o f t h e i n c u b a t i o n m i x t u r e w a s 1 1 0 pl. S u b s e q u e n t l y , s t a p h y l o c o c c i n 1580 (250 arbitrary units per ml) or only b u f f e r were added and 2 rain later the incubat i o n m i x t u r e s w e r e r a p i d l y d i l u t e d 5 0 - f o l d w i t h p r e w a r m e d i n c u b a t i o n b u f f e r , c o n t a i n i n g e i t h e r 5 rnM or n o N A D H , a n d [ 3 H ] i n u h n ( 0 . 5 ~ C i / m l ) . S a m p l e s (1 m l ) w e r e t a k e n at v a r i o u s t i m e i n t e r v a l s a n d f i l t e r e d w i t h o u t f u r t h e r w a s h i n g . T h e a m o u n t o f e x t r a v e s i c u l a r w a t e r r e m a i n i n g on t h e f i l t e r w a s c a l c u l a t e d f r o m t h e 3 H r a d i o a c t i v i t y . T h e r e s u l t s are e x p r e s s e d as p e r c e n t a g e L - g l u t a m a t e r e m a i n i n g i n s t a p h y l o e o c c i n t r e a t e d or u n t r e a t e d v e s i c l e s a f t e r d i l u t i o n , w i t h r e g a r d t o t h e L - g l u t a m a t e c o n c e n t r a t i o n in v e s i c l e s t h a t w e r e n o t d i l u t e d . S y m b o l s : o, c o n t r o l s d i l u t e d in m e d i u m c o n t a i n i n g 5 m M N A D H ; ~, s t a p h y l o c o c c i n 1 5 8 0 t r e a t e d , d i l u t e d in m e d i u m c o n t a i n i n g 5 m M N A D H ; e, c o n t r o l s , d U u t e d in m e d i u m w i t h o u t N A D H ( r e s u l t i n g in a final c o n c e n t r a t i o n o f 0.1 m M ; A s t a p h y l o c o c c i n 1 5 8 0 t r e a t e d , d i l u t e d in m e d i u m w i t h o u t N A D H ( f i n a l c o n c e n t r a t i o n 0.1 m M ) .
394
% RESIDUAL GLUTAMATE I0C
80
60
~A /.0
20
I
I
I
0
1
2
I
I
3 /-. TIME [mini
I 5
F i g . 5. E f f e c t o f s t a p h y l o c o c c i n 1 5 8 0 o n L - g l u t a m a t e e x c h a n g e . T h e c o n d i t i o n s w e r e t h e s a m e as in F i g . 4, e x c e p t t h a t t h e v e s i c l e s w e r e d i l u t e d 5 0 - f o l d in a b u f f e r m e d i u m c o n t a i n i n g 5 m M N A D H a n d 2 m M u n labelled L-glutamate. Symbols: e, vesicles not treated with staphylococcin 1580; A treated with 100 arbitrary units per ml; D treated with 250 arbitrary units per ml.
The results show that staphylococcin 1580 has a slight effect on the L-glutamate carrier function, either directly affecting the catalytic properties or uncoupling the carrier from the energy supply.
Effect o f staphylococcin 1580 on 3,3'-dipropylthiadicarbocyanine fluorescence Recently, Bhattacharryya et al. [ 12] used the fluorescent dye 3,3'-dipropylthiadicarboeyanine to m o n i t o r the membrane potential in membrane vesicles of A. vinelandii. The fluorescence quench can be directly correlated to the membrane potential (in mV) by calibration with the diffusion potential posed on potassium-loaded vesicles upon addition of valinomycin. Our results (not shown) obtained with membrane vesicles of B. subtilis were similar to those reported by Bhattacharryya et al. [12]. Fig. 6 shows that vesicles quenched the 3,3'-dipropylthiadicarbocyanine fluorescence upon addition of NADH (steady state 99 mV). Addition of staphylococcin 1580 instantaneously abolished the fluorescence quench to a final level with depends on the a m o u n t of bacteriocin applied; the resulting membrane potential was 79 and 40 mV after treatment with 500 and 2500 arbitrary units of staphylococcin 1580 per mg membrane protein, respectively. Staphylococcin 1580 abolishes also the membrane potential built up by the diffusion potential of K ÷ (Fig. 7). In the presence of the bacteriocin a lower optimum was reached and the quench was dissipated more rapidly. Almost no influence of staphylococcin 1580 was observed on the 3,3'-dipropylthiadicarbocyanine fluorescence with "resting" vesicles irrespective of sodium- or potassium-loaded vesicles were used in the presence of either
395 FLUORESCENCE [arbitrary units1
FLUORESCENCE]arbffrery units]
80
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I
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1
2
3
z. TIME [rain]
5
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l. TIME [mini
Fig. 6. E f f e c t of s t a p h y l o c o c c i n 1 5 8 0 o n t h e r e s p i r a t i o n i n d u c e d q u e n c h i n g o f 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e (DiS-C3) f l u o r e s c e n c e . M e m b r a n e vesicles ( 2 0 p g p r o t e i n / m l ) w e r e i n c u b a t e d at 2 5 ° C in 50 m M p o t a s s i u m p h o s p h a t e ( p H 6.6), 10 m M MgSO 4 a n d 0.8 p M 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e . R e s p i r a t i o n was s t a r t e d b y t h e a d d i t i o n of 5 m M N A D H . W h e n t h e f l u o r e s c e n c e q u e n c h r e a c h e d a p l a t e a u , s t a p h y l o c o c c i n 1 5 8 0 in a final c o n c e n t r a t i o n of 2 5 0 0 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ( d o t t e d line), o r 500 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ( b r o k e n line), or only b u f f e r (solid line) w a s a d d e d . A t t h e e n d o f the e x p e r i m e n t g r a m i c i d i n D (1 # g / m l ) was a d d e d , w h i c h c o m p l e t e l y abolishes t h e m e m b r a n e potential. Fig. 7. E f f e c t of s t a p h y l o c o c c i n 1 5 8 0 o n t h e 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e (DiS-C 2 - f l u o r e s c e n c e q u e n c h i n g , i n d u c e d b y a p o t a s s i u m d i f f u s i o n p o t e n t i a l . P o t a s s i u m - l o a d e d m e m b r a n e vesicles ( 2 0 pg protein p e r m l ) w e r e i n c u b a t e d f o r 2 m i n at 25°C in a b u f f e r c o n t a i n i n g 49 m M s o d i u m p h o s p h a t e a n d 1 m M p o t a s s i u m p h o s p h a t e ( p H 6 . 6 ) , 10 m M M g S O 4 , a n d s t a p h y l o c o c c i n 1 5 8 0 in a final c o n c e n t r a t i o n o f 2 5 0 0 ( d o t t e d line) a n d 500 a r b i t r a r y u n i t s p e r m g m e m b r a n e p r o t e i n ( b r o k e n line) o r b u f f e r i n s t e a d o f t h e bact e r i o c i n (solid line). T h e n 3 , 3 ' - d i p r o p y l t h i a d i c a r b o c y a n i n e (0.8 #M) was a d d e d , f o l l o w e d a b o u t 1 m i n l a t e r b y v a l i n o m y c i n (0.5/~M). A t t h e e n d o f the e x p e r i m e n t g r a m i e i d i n D (1 p g / m l ) was a d d e d .
sodium or potassium phosphate buffer. T he refore staphylococcin 1580 cann o t be regarded as a specific c o n d u c t o r of either K ÷ or Na ÷.
Discussion Many observations have poi nt e d to the cytoplasmic m e m b r a n e as the target for the action o f staphylococcin 1580 [2,3,5]. T h e r e f o r e a n u m b e r of effects o f the bacteriocin on isolated m e m b r a n e vesicles of B. subtilis were studied and c o m p a r e d to the effects on whole cells. Konings [11,12] has shown t hat the m e m b r a n e o f B. subtilis W23 vesicles, pr e pa red by the m e t h o d used in this study, are topologically identical to the membranes of intact cells. Moreover, the specific transport rates of these vesicles approxi m at e those of intact cells when expressed as mol t r a ns por t e d per weight unit of m e m b r a n e protein. However, some structural and functional differences between cells and m em brane vesicles affect their susceptibility to staphylococcin 1580. The access of the bacteriocin to the m e m b r a n e could be impeded in whole cells due t o the pres-
396 ence of cell wall aspecific binding sites for the bacteriocin [22]. Moreover, the complex balance between intra- and extracellular solutes in whole cells may result in differences in the sensitivity to the bacteriocin on comparing whole cells with m e m b r a n e vesicles. This may be illustrated by the suggestion of Brewer [24] th at after colicin K t r e a t m e n t the membrane potential of E. coli cells depends on an electrochemical potential of an anion. In accordance, anions, such as pyruvate and p h o s p h o r y l a t e d intermediates of glycolysis, are lost after colicin E1 or K t r e a t m e n t [25]. The results obtained in this investigation allow a classification of the effects of staphylococcin 1580 into at least three groups (Table II). The anaerobic influx o f L-glutamate in m em br ane vesicles is most sensitive to the bacteriocin. At present, the way in which staphylococcin 1580 affects this influx is not clear. The kinetics of solute permeation has been extensively studied with m e mb r an e vesicles of E. coli [26,27] and S. aureus [28]. In S. aureus vesicles serine efflux, and influx under energized conditions, exhibited saturation kinetics. However, anaerobic influx appeared to be a non-saturable process, and it was co n clu d ed that the transport carriers catalyze facilitated diffusion in the direction of efflux only [28]. Extending this conclusion to L-glutamate influx in B. subtilis vesicles would mean that staphylococcin 1580 specifically blocks this passive diffusion. Such an effect could be explained by a change in membrane charge or h y d r o p h o b i c i t y of the diffusion barrier. A second group of effects are exerted by staphylococcin 1580 when applied to cells or m e m b r a n e vesicles in concentrations of about 250 and 4 0 0 - - ! 6 0 0 arbitrary units per mg of m e m b r a n e protein, respectively. These effects can be explained by a dissipation of the p r o t o n motive force since A~ (rubidium uptake in the presence of valinomycin; 3,3'-dipropylthiadicarbocyanine fluorescence) and ApH (acetate uptake) of m em br ane vesicles are abolished by these bacteriocin concentrations. The observed differences between the inhibitory effects on individual t r ans por t systems probably result from (i) a different sensing of the transport carriers to the energy supply, and (ii) a difference in direct susceptibility of the carrier to the bacteriocin, since it was shown to affect the L-glutamate carrier. Whole cells were shown to be a b o u t five times m ore sensitive to staphylococcin 1580 than vesicles, when the effect of the bacterio-
T A B L E II CLASSIFICATION OF EFFECTS AND MEMBRANE VESICLES
INDUCED BY STAPHYLOCOCCIN
1 5 8 0 IN B. S U B T 1 L I S
CELLS
System
Process
Amount of bacteriocin yielding a half-maximal effect (arbitrary units/ mg membrane protein)
Vesicles Vesicles
A n a e r o b i c L - g l u t a m a t e i n f l u x , s t e a d y - s t a t e level A c t i v e u p t a k e o f s o l u t e s c o u p l e d to t h e h i g h - e n e r g y s t a t e o f the membrane * ; membrane potential (fluorescence); L-glutamate efflux L - G l u t a m a t e u p t a k e ; killing a-Methylglucoside uptake
20---60 400---1600
Cells Cells
* S o l u t e s listed in T a b l e I.
250 9400
397
cin on the uptake of L-glutamate was measured. This may reflect the complex composition of the membrane potential in whole cells. Inhibition of ~-methylglucoside uptake in B. subtilis cells required the application of distinctly higher concentrations of staphylococcin 1580. This seems to contradict the previously reported inhibition of o-nitrophenyl-~-galactoside hydrolysis in S. aureus, which is also mediated by the p h o s p h o e n o l p y r u v a t e phosphotransferase system [29], by moderate staphylococcin concentrations. However, it may reflect a different regulation of uptake systems in both organisms, rather than the induction of gross permeability changes in S. aureus. Moreover, the present results are in accordance with the observations made with colicins E1 and K [18] and Ia [19]. References 1 L u r i a , S.E. ( 1 9 7 3 ) In B a c t e r i a l M e m b r a n e s a n d Walls (Leive, L., ed.), p p . 2 9 3 - - 3 3 0 , M a r c e l D e k k e r , Inc., New York 2 J e t t e n , A.M. a n d V o g e l s , G . D . ( 1 9 7 2 ) A n t i m i c r o b . A g e n t s C h e m o t h e r . 2, 4 5 6 - - 4 6 3 3 J e t t e n , A.M. a n d V o g e l s , G . D . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 1 , 4 8 3 - - 4 9 5 4 W e e r k a m p , A., G e e r t s , W. a n d V o g e l s , G . D . ( 1 9 7 7 ) A n t i m i e r o b . A g e n t s C h e m o t h e r . 1 2 , 3 1 4 - - 3 2 1 5 W e e r k a m p , A. a n d V o g e l s , G . D . ( 1 9 7 8 ) A n t i m i c r o b . A g e n t s C h e m o t h e r . , in t h e p r e s s 6 B h a t t a c h a r r y y a , P., W e n d t , L., W h i t n e y , E. a n d Silver, S. ( 1 9 7 0 ) S c i e n c e 1 6 8 , 9 9 8 - - 1 0 0 0 7 B i s s c h o p , A., D o d d e m a , H. a n d K o n i n g s , W.N. ( 1 9 7 5 ) J. B a c t e r i o l . 1 2 4 , 6 1 3 - - 6 2 2 8 K o n i n g s , W . N . , B i s s c h o p , A. a n d D a a t s e l a a r , M.C.C. ( 1 9 7 2 ) F E B S L e t t . 2 4 , 2 6 0 - - 2 6 4 9 M a t i n , A. a n d K o n i n g s , W.N. ( 1 9 7 3 ) E u r . J. B i o c h e m . 3 4 , 5 8 - - 6 7 1 0 F o r t n a g e l , P. a n d F r e e s e , E. ( 1 9 6 8 ) J. B a c t e r i o l . 9 5 , 1 4 3 1 - - 1 4 3 8 11 K o n i n g s , W . N . , B i s s c h o p , A . , V e e n h u i s , M. a n d V e r m e u l e n , C . A . ( 1 9 7 3 ) J. B a c t e r i o l . 1 1 6 , 1 4 5 6 - 1465 12 B h a t t a e h a r r y y a , P., S h a p i r o , S.A. a n d B a r n e s , J r . , E.M. ( 1 9 7 7 ) J. B a c t e r i o l . 1 2 9 , 7 5 6 - - 7 6 2 1 3 W a g g o n e r , A. ( 1 9 7 6 ) J. M e m b r a n e Biol. 2 7 , 3 1 7 - - 3 3 4 1 4 J e t t e n , A . M . , V o g e l s , G . D . a n d d e W i n d t , F. ( 1 9 7 2 ) J. B a c t e r i o l . 1 1 2 , 2 3 5 - - 2 4 2 1 5 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 1 6 K a b a c k , H . R . ( 1 9 7 1 ) In M e t h o d s in E n z y m o l o g y ( J a c o b y , W.B., ed.), V o l . 2 2 , p p . 9 9 - - 1 2 0 , A c a d e m i c Press, N e w Y o r k 17 D e l o b b e , A., H a g u e n a u e r , R. a n d R a p o p o r t , G. ( 1 9 7 1 ) B i o c h i m i e 5 3 , 1 0 1 5 - - 1 0 2 1 1 8 J e t t e n , A.M. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 4 0 , 4 0 3 - - 4 1 1 1 9 G i l c h r i s t , M. a n d K o n i s k y , J. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 2 4 5 7 - - 2 4 6 2 2 0 R a m o s , S., S e h u l d i n e r , S. a n d K a b a c k , H . R . ( 1 9 7 6 ) P r o c . N a t l . A c a d . Sci. U.S. 7 3 , 1 8 9 2 - - 2 8 9 6 21 K o n i n g s , W.N. ( 1 9 7 5 ) A r c h . B i o c h e m . B i o p h y s . 1 6 7 , 5 7 0 - 5 8 0 2 2 J e t t e n , A.M. a n d Vogels, G . D . ( 1 9 7 2 ) J. B a c t e r i o l . 1 1 2 , 2 4 3 - - 2 5 0 2 3 B i s s e h o p , A., B o o n s t r a , J., S i p s , H . J . a n d K o n i n g s , W.N. ( 1 9 7 5 ) F E B S L e t t . 6 0 , 1 1 - - 1 6 24 Brewer, G.J. (1976) Biochemistry 15, 1387--1392 2 5 Fields, K . L . a n d L u r i a , S.E. ( 1 9 6 9 ) J. B a c t e r i o l . 9 7 , 6 4 - - 7 7 2 6 K a b a e k , H . R . a n d B a r n e s , J r . , E.M. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 5 5 2 3 - - 5 5 3 1 27 L o m b a r d i , F . J . a n d K a b a c k , H . R . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 8 4 4 - - 7 8 5 7 28 S h o r t , S . A . a n d K a b a c k , H . R . ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 4 2 7 5 - 4 2 8 1 29 K e n n e d y , E.P. a n d S c a r b o r o u g h , G . A . ( 1 9 6 7 ) P r o c . N a t l . A c a d . Sci. U.S. 5 8 , 2 2 5 - - 2 2 8
