On the relationship between the nonlinear dielectric properties and respiratory activity of the obligately aerobic bacterium Micrococcus luteus

423

Bioelectrochemistry and Bioenergetics, 26 (1991) 423-439 A section o f I. Eiectroanal. Chem., and constituting Vol. 321 (1991) Elsevier Sequoia S.A., Lausanne

JEC BB 01418

On the relationship between the nonlinear dielectric properties and respiratory activity of the obligately aerobic bacterium Micrococcus luteus A n d r e w M. W o o d w a r d a n d D o u g l a s B. K e l i * Departmeut of Biological Sciences, Universiw College of Wales, Aberystwyth, Dyfed SY23 3DA (UIO (Received 3 July 1991) Abstract We have used our previously-described, four-terminal, nonlinear dielectric spectrometer to study the generation o f nonlinear dielectricity by th~ obligately aerobic, heterotrophic, respiratory bacterium Micrococcus luteus. Intact cells of M. lureus generate harmonics when excited with single sinnsoids o f appropriate voltage and frequency. Two frequency windows are observed, respectively below ("low , frequencies") and above ("high frequencies") ca 100 Hz. T h e low-frequency region shows a voltage window o f some ± 0 . 4 - 1 . 6 V (zero-to-peak, as judged on the o u t e r electrodes). Anaerobic (non-respiring) cells generate odd- num be r ed harmonics, whilst aerobic (respiring) ceils generate even-numbered harmonics. In the high-frequency range, intact cells o f M. luteus (and also o f Saccharomyces cereuisiae) generate both odd- and even-numbered harmonics, whether aerobic o r anaerobic. In the low-frequency range, the inhibition o f respiration by cyanide o r by H Q N O (2-n-heptyl-4-hydroxyquinoline-N-c~jde) causes the appearance o f o d d - n u m b e r e d harmonics in aerobic cell suspensions. H i g h e r concentrations o f cyanide (but not of H Q N O ) cause the disappearance o f all harmonics. T h e s e facts are consistent with the known effects of these inhibitors on electron transport in the branched respiratory chain o f this organism, and suggest that the major source of nonlinear dielectriciw within the respiratory chain is either in the branch to cytochrome bs5 s o r proximal to the site at which H Q N O binds. M e m b r a n e vesicles o f M. luteus generate harmonics with patterns similar to those found in intact c~lls. In the low-frequency region, the addition o f N A D H to the m e m b r a n e vesicles causes the replacement of odd-numbered harmonics by even-numbered harmonics, providing extremely clear-cut evidence that the type o f harmonics observed depends strongly on w h e t h e r or not the cells are respiring. In the high-frequency region, both odd- and even-numbered h a r m o n i c s a r e generated by m e m b r a n e vesicles, w h eth er o r not the membranes are carrying out electron transport. Liposomes consisting o f pu re phospholipids show similar patterns of harmonics, suggesting that the high-frequency nonlinear dielectricity may be ascribed to the lipid portion o f the membranes o f cells and vesicles. By contrast, tL: expression o f nonlinear dielectriciW at low exciting frequencies is determined by the respiratory system, and wh eth er o r not it is active. This p h e n o m e n o n provides a novel m et hod for the m e a s u r e m e n t o f respiratory activity in situ.

* T o whom correspondence should be addressed. 0302-4598/91/$03.50 © 1991 - Elsevier Sequoia S.A. All fights reserved

424 INTRODUCTION

W e have recently d e m o n s t r a t e d t h e g e n e r a t i o n o f n o n l i n e a r dielectrieity by yeast cell suspensions [1-3], m a n i f e s t as h a r m o n i e s or b e a t f r e q u e n c i e s w h e n cells were excited, within a p p r o p r i a t e voltage a n d f r e q u e n c y windows, by sinusoidally m o d u l a t e d electric fields. In this system, i n h i b i t o r studies revealed t h a t t h e m a j o r source of the n o n l i n e a r dielectrieity observed was the H + - A T P a s e p r e s e n t in t h e p l a s m a m e m b r a n e of this organism. W h e n t h e exciting field was a single sinusoid of a p p r o p r i a t e frequency, t h e type o f h a r m o n i c s observed d e p e n d e d u p o n w h e t h e r t h e cells were resting or glycolysing: in t h e f o H a e r ease, o d d - n u m b e r e d h a r m o n i c s w e r e observed, whilst cells which w e r e actually m e t a b o l i s i n g glucose a n d g e n e r a t ing A T P via substrate-level p h o s p h o r y l a t i o n g e n e r a t e d e v e n - n u m b e r e d h a r m o n i c s [1]. This type o f b e h a v i o u r could be a c c o u n t e d for in terms o f a simple d o u b l e p o t e n t i a l well model, and t h e f r e q u e n c y o p t i m u m observed, ca 1 5 - 2 0 Hz, was strikingly similar to the kea t of t h e yeast H + - A T P a s e [2]. Electrical fields of low f r e q u e n c y , i.e. o f a f r e q u e n c y low with respect to t h e characteristic frequency (ca. 1 M H z ) o f t h e /3-dielectric dispersion o f cells a n d tissues (mainly r e p r e s e n t i n g t h e c h a r g i n g o f a static m e m b r a n e c a p a c i t a n c e [4-15]) do n o t effectively p e n e t r a t e t h e cell m e m b r a n e . Thus, and whilst this p h e n o m e n o n u n d e r p i n s the fact t h a t t h e n o n l i n e a r dielectric m e t h o d is selective for m e m b r a n o u s enzymes in intact cells, t h e r e could b e no c o n t r i b u t i o n o f r e s p i r a t o r y c h a i n enzymes, which are located in t h e m i t o c h o n d r i a o f t h e s e e u k a r y o t i c (yeast) cells, to t h e n o n l i n e a r dielectric spectra m e a s u r e d . By contrast, o f course, t h e r e s p i r a t o r y c h a i n of p r o k a r y o t e s /s f o u n d in t h e i r cytoplasmic m e m b r a n e . It was t h e r e f o r e o f g r e a t i n t e r e s t to extend t h e n o n l i n e a r dielectric spectroscopic a p p r o a c h to a suitable h e t e r o t r o p h i c r e s p i r a t o r y b a c t e r i u m , so as to d e t e r m i n e w h e t h e r , in such a n organism, t h e r e might be a c o n t r i b u t i o n to the n o n l i n e a r dielectric s p e c t r a f r o m r e s p i r a t o r y c h a i n enzymes. T h e p r e s e n t article describes such e x p e r i m e n t s using t h e obligately aerobic soil b a c t e r i u m Micrococcus luteus, at t h e level o f b o t h t h e intact cell a n d t h e m e m b r a n e vesicle. It was found, for the first time, t h a t t h e r e s p i r a t o r y c h a i n was i n d e e d t h e m a j o r source of n o n l i n e a r dielectricity in this organism, a n d t h a t the type o f h a r m o n i c s observed ( o d d - n u m b e r e d vs. e v e n - n u m b e r e d ) d e p e n d e d u p o n w h e t h e r or n o t t h e r e s p i r a t o r y c h a i n was active, a f i n d i n g c o m p l e m e n t i n g t h a t observed for t h e H + - A T P a s e in yeast. I n a d d i t i o n , we observed t h e g e n e r a t i o n o f n o n l i n e a r dieleetricity in the kHz r a n g e in cells o f b o t h y e a s t a n d M. luteus; since we also observed this in m e m b r a n e vesicles a n d in liposomes consisting purely o f p h o s p h o l i p i d , it is ascribed to t h e c o n f o r m a t i o n a l a n d c o n f i g u r a t i o n a l dynamics o f t h e lipid p o r t i o n of t h e m e m b r a n e s a n d m e m b r a n e vesicles studied. EXPERIMENTAL

Biological material. Micrococcus luteus (syn. M. lysodeikticus) was g e n e r a l l y o b t a i n e d as a f r e e z e - d r i e d s u s p e n s i o n f r o m the Sigma C h e m i c a l C o m p a n y , Poole,

Dorset. It was r e h y d r a t e d in a b u f f e r consisting o f 50 m M K H 2 P O 4 , 1 m M MgSO4, p H 7.0. In some eases, Micrococcus luteus F l e m i n g strain 2665 was used, a n d was m a i n t a i n e d o n Difco n u t r i e n t a g a r a n d grown aerobically as described e l s e w h e r e [16]. N o substantive differences were observed in t h e n o n l i n e a r dielectric beh a v i o u r o f t h e two strains. M e m b r a n e vesicles were p r e p a r e d by o s m o t l e t r e a t m e n t o f p r o t o p l a s t s c r e a t e d by t r e a t m e n t o f intact eeU suspensions with lyaozyme. F o r this, 0.5 g dry wt. o f cells w e r e s u s p e n d e d in 10 ml o f t h e b u f f e r described above, to w h i c h were a d d e d 1.5 mg lysozyrne. T h e s e were i n c u b a t e d at 20°C for 10 minutes. T h e n , a crystal o f D N A a s e was a d d e d , a n d t h e m e m b r a n e s collected by centrifugation a n d r e s u s p e n d e d in 10 ml o f t h e original buffer. C o n c e n t r a t i o n s o f m e m b r a n e s a r e . q u o t e d in terms o f t h e e q u i v a l e n t dry weight of cells from which they were prepared. Liposomes w e r e p r e p a r e d via t h e e h o l a t e dialysis p r o c e d n r e . 2 g o f c r u d e s o y b e a n L-c~-phosphatidylcholine (Sigma Woe IV-S) p l u s 0.8 g s o d i u m c h o l a t e w e r e a d d e d to 20 ml o f t h e p h o s p h a t e b u f f e r d e s c r i b e d above, a n d s o n i c a t e d in bursts (3 × 1 rain cumulative at t h e full p o w e r o f an M S E u l t r a s o n i c a t o r ) until t h e mixture h a d dissolved. T h e c h o l a t e p h o s p h o l i p i d mixture was dialysed o v e r n i g h t at 4°C vs. p h o s p h a t e b u f f e r a n d t h e liposomes s t o r e d at 4"C a n d used w i t h i n 3 days. Nonlinear dielectric rne~,~surements were p e r f o r m e d exactly as described previously [1], save t h a t t h e oscillator used was a K r o h n - H i t e m o d e l 4400A ultra-lownoise g e n e r a t o r [2]. T h e powers in d i f f e r e n c e spectra are given to t h e n e a r e s t dB. Chemicals. All o f t h e s e w e r e o b t a i n e d f r o m t h e Sigma C h e m i c a l C o m p a n y o r B D H Ltd, Poole, Dorset. W a t e r was singly-distilled in an all-glass a p p a r a t u s . RESULTS

F i g u r e 1 shows a typical n o n l i n e a r dielectric s p e c t r u m o f a cell s u s p e n s i o n (25 m g dry wt m l - 1 ) o f M. luteus (Fig. 1A), a s u p e r n a t a n t w h o s e conductivity was a d j u s t e d t o m a t c h t h a t o f t h e cell s u s p e n s i o n (Fig. 1B), a n d t h e i r ratio (Fig. 1C). T h e exciting f r e q u e n c y was 30 I-lz a n d t h e field strength, as j u d g e d by m e a s u r e m e n t s b e t w e e n t h e o u t e r e l e c t r o d e s was ___0.7 V e r a - t (zero-to-peak). It m a y b e o b s e r v e d that, at this cell c o n c e n t r a t i o n , t h e s p e c t r u m c o n t a i n s b o t h o d d - a n d e v e n - n u m b e r e d h a r m o n i e s . M. luteus is a n obligate a e r o b e , b e i n g u n a b l e to f e r m e n t , a n d since n o atterhpt was m a d e to oxygenate t h e cells, t h e b u l k o f a cell s u s p e n s i o n o f this cell density in o u r r e a c t i o n vessel w o u l d have b e c o m e a n a e r o b i c d u r i n g t h e incub~,tion p h a s e p r i o r t o t a k i n g t h e s p e c t r u m ( a l t h o u g h t h e t o p will have b e e n aerobic). T o e l a b o r a t e this point, a n d to c h e e k t h a t t h e g e n e r a t i o n o f h a r m o n i e s was i n d e e d caused by t h e cells, Fig. 2 shows t h e d e p e n d e n c e o n cell c o n c e n t r a t i o n o f t h e n o n l i n e a r dielectric s p e c t r u m o f intact cell suspensions o f M. iuteus. It m a y be observed that, a b o v e a c o n c e n t r a t i o n o f some 25 m g / r a l , t h e m a g n i t u d e of t h e o d d - n u m b e r e d (third) h a r m o n i c d e p e n d s m o n o t o n i c a l l y o n t h e cell c o n c e n t r a t i o n . However, b e l o w 25 m g dry wt. c e l l s / m l , t h e c~-dd-numbered h a r m o n i c disappe.qrs a n d is r e p l a c e d by an e v e n - n u m b e r e d one, strongly suggestive t h a t u n d e r t h e s e c o n d i t i o n s t h e cells b e c o m e a e r o b i c a n d g e n e r a t e e v e n - n u m b e r e d

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Frequency/Hz Fig. 1. N o n l i n e a r dielectric p r o p e r t i e s o f M. luteus. A s u s p e n s i o n o f cells (25 m g dry wt. m l - i, p r e p a r e d as d e s c r i b e d in the -Experimental section), w a s p l a c e d in t h e test ceil, a n d a s a m p l e o f its s u p e r n a t a n t (with its conductivity at 1 k H z a d j u s t e d to m a t c h t h a t o f the cells) was p l a c e d in t h e r e f e r e n c e cell. S p e c t r a w e r e o b t a i n e d using an exciting v o l t a g e o f 5= 0.7 V z e r o - t o - p e a k (field s t r e n g t h + 1.05 V c m - 1 as m e a s u r e d b e t w e e n the o u t e r e l e c t r o d e s , + 130 m V c m - t as m e a s u r e d b e t w e e n the i n n e r e l e c t r o d e s ) at a f r e q u e n c y o f 30 H z , a n d e a c h s p e c t r u m r e p r e s e n t s t h e a v e r a g e o f 30 blocks. ( A ) T e s t cell. (B) R e f e r e n c e ceil• (C) T e s t cell m i n u s r e f e r e n c e cell.

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Fig. -3. E f f e c t o f r e s p i r a t o r y chain activity o n t h e n o n l i n e a r dielectric b e h a v i o u r o f intact cells o f M. luteus. M e a s u r e m e n t s w e r e m a d e as d e s c r i b e d in t h e l e g e n d t o Fig. 2, at a n exciting f r e q u e n c y o f 10 H z a n d a voltage o f =t=0.8 V ( z e r o - t o - p e a k ) , at a cell c o n c e n t r a t i o n o f 6 g 1-1. ( A ) A e r o b i c cells. (B) A n a e r o b i c cells.

h a r m o n i c s via activation o f t h e i r r e s p i r a t o r y chains. This was c o n f i r m e d by a n e x p e r i m e n t in which we c o m p a r e d cells at a c o n c e n t r e t i o n o f 6 g / l u n d e r explicitly aerobic a n d a n a e r o b i c c o n d i t i o n s (Fig. 3), t h e l a t t e r e n f o r c e d by filling t h e r e a c t i o n vessel completely, closing it to the ingress o f a t m o s p h e r i c oxyge:n a n d waiting f o r t h e cells to use u p t h e residual oxygen. O n a day-to-day basis, the c o n c e n t r a t i o n at which t h e cells b e c a m e a n a e r o b i c u n d e r r o u t i n e c o n d i t i o n s was r a t h e r variable (as may be gleaned, for instance, from Figs. 1 a n d 2), p r e s u m a b l y reflecting variations in t h e e n d o g e n o u s r e s p i r a t o r y rate a n d t e m p e r a t u r e o f m e a s u r e m e n t , b u t was n o r m a l l y in t h e r a n g e 10-25 mg dry wt. m l - 1 W e m a y m e n t i o n h e r e t h a t c o n t r o l e x p e r i m e n t s (lacking eel;s) i n d i c a t e d that 0 2 p e r se h a d n o effect o n t h e generation o f n o n l i n e a r dielectricity. Since t h e g e n e r a t i o n o f n o n l i n e a r dielectricity by yeast eell~ s h o w e d a r a t h e r n a r r o w voltage a n d f r e q u e n c y window [1-3], we s t u d i e d w h e t h e r this m i g h t also be true of M. luteus. Figures 4 a n d 5 show, respectively, t h a t within t h e voltage a n d frequency r a n g e indicated, windows are i n d e e d manifest. T h e s e e x p e r i m e n t s were

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carried o u t at a cell c o n c e n t r a t i o n o f 10 mg dry wt. m l - t , w h e r e b o t h odd- and e v e n - n u m b e r e d h a r m o n i c s were present; it may be observed t h a t these have r a t h e r similar voltage (Fig. 4) and frequency (Fig. 5) windows. In the e x p e r i m e n t s with yeast ceils [1-3], we did not study in any detail the effects of exciting frequency b e y o n d ca. 100 Hz, since n o n e were observed within the voltage window used at the lower frequencies. However, in the p r e s e n t case, when we extended the exciting frequency to values significantly b e y o n d 100 Hz, a second region of h a r m o n i c g e n e r a t i o n was observed (Fig. 6), with b o t h odd- a n d e v e n - n u m b e r e d h a r m o n i c s being g e n e r a t e d . T h e frequency- a n d voltage-dependences are shown, respectively, in Figs. 7 a n d 8, w h e r e it may be observed that t h e f i e l d - d e p e n d e n c e is r a t h e r d i f f e r e n t (Fig. 8) from t h a t observed in the low-frequency region (Fig. 4). T h e s e spectra did not c h a n g e substantially as a f u n c t i o n of w h e t h e r t h e cells were aerobic or a n a e r o b i c (not shown). However, they did d e p e n d u p o n the p r e s e n c e of ceils, as shown in the ceil c o n c e n t r a t i o n - d e p e n d e n c e (Fig. 9). M. l u t e u s , in c o m m o n with m a n y o t h e r bacteria [17,18], possesses a b r a n c h e d respiratory chain [19], cytochromes b558 (probably a c y t o c b r o m e o) a n d a a 3 acting as t e i m i n a l electron acceptors [20-22]. Low c o n c e n t r a t i o n s o f t h e respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide ( H Q N O ) can act partially to inhibit respiration at the b c y t o c h r o m e level (after t h e branch), whilst cyanide inhibits respiration via b o t h c y t o e h r o m e a a 3 (at " l o w " c o n c e n t r a t i o n s ) a n d CYt o c h r o m e o (at high concentrations); indeed, l o w c o n c e n t r a t i o n s o f cyanide can actually s t i m u l a t e the overall respiratory rate by diverting t h e flux o f electrons from the well-coupled m a i n chain to t h e m o r e poorly c o u p l e d alternative b r a n c h [22]. It was thus o f interest to study t h e effects of these inhibitors on the g e n e r a t i o n o f n o n l i n e a r dieleetriciL" by M . l u t e u s ceils. Figure 10 shows the effect of H Q N O on t h e g e n e r a t i o n of h a r m o n i e s by aerobic cells of M . l u t e u s . It may be observed t h a t in t h e absence o f t h e inhibitor t h e r e are very few o d d - n u m b e r e d harmonics, but t h a t increasing c o n c e n t r a t i o n s o f t h e inhibitor cause t h e m to appear. T h a t inhibition o f t h e h a r m o n i c s is n o t observed

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m i g h t be c o n s i d e r e d to b e c o n s i s t e n t w i t h t h e i n a b i l i t y o f this m o l e c u l e a l o n e to i n h i b i t fully r e s p i r a t i o n in this o r g a n i s m [22]. H o w e v e r , t h e f a c t t h a t H Q N O actually causes a substantial appearance of o d d - n u m b e r e d harmonics rather sugg e s t s t h a t t h e p a r t o f t h e r e s p i r a t o r y c h a i n b e i n g o b s e r v e d is n o t l o c a t e d at t h e site 10

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" ~

.'

560

1000

1500

2000

Frequency/llz Fig. 7. Effect o f f r e q u e n c y o n the n o n l i n e a r dielectric p r o p e r t i e s o f intact cell s u s p e n s i o n s o f . M . luteus. M e a s u r e m e n t s w e r e m a d e exactly ~:~ d e s c r i b e d in the l e g e n d to Fig. 6(C), save t h a t t h e f r e q u e n c y ( a b o v e 100 H z ) was v a r i e d as indicated.

430 •

.E

•

•

i

•

•

•

•

|

"

'

"

•

I

•

•

•

•

o

e--

~

-20 0

0.5

1

1.5

VoLtage(zero-fo-peak}/V

2

Fig. 8. E f f e c t o f voltage o n t h e n o n l i n e a r dielectric p r o p e r t i e s o f intact cell s u s p e n s i o n s o f M• luteus. M e a s u r e m e n t s w e r e m a d e exactly as described in t h e legends to Figs• 6 a n d 7, at a f r e q u e n c y o f 1 k H z a n d a cell c o n c e n t r a t i o n o f 25 g 1 - t , save t h a t t h e v o l t a g e ( z e r o - t o - p e a k ) was v a r i e d as indicated•

0 "o~

-10

E

-15

: =_

-20

"~

-25

=-

-30

0.1

1

10

Ceil c o n c e n t r a t i o n / r a g

100 d r y wt. rn1-1

Fig. 9. E f f e c t o f cell c o n c e n t r a t i o n o n t h e n o n l i n e a r dielectric p r o p e r t i e s o f intact cell s u s p e n s i o n s o f M. luteus. M e a s u r e m e n t s w e r e m a d e exactly as d e s c r i b e d in the l e g e n d s to Figs• 6 - 8 , save t h a t t h e exciting f r e q u e n c y w a s 1 k H z , t h e v o l t a g e o n t h e o u t e r e l e c t r o d e s ( z e r o - t o - p e a k ) w a s :i:0.7 V , a n d t h e cell c o n c e n t r a t i o n w a s v a r i e d a s indicated.

E

"=-=_~o 2 0 -40 0

2

4

6

8

10

[HqNo]/ram Fig. 10. Effect o f Measurements were 3 g ! - ! an exciting HQNO was present

•

.-.r

,

H Q N O on t h e n o n l i n e a r dielectric p r o p e r t i e s o f a e r o b i c cells o f M. luteus. p e r f o r m e d exactly as d e s c r i b e d in t h e l e g e n d to Fig• 2, at a cell c o n c e n t r a t i o n o f f r e q u e n c y o f 30 H z , a n d an exciting voltage ( z e r o - t o - p e a k ) o f :J= I V, save t h a t at t h e c o n c e n t r a t i o n s indicated•

431

20

"~o

10

s .~

0

~-

-10 0

5

10

15

20

IKCN!/ram Fig. 11. Effect o f c y a n i d e o n the n o n l i n e a r dielectric p r o p e r t i e s o f a e r o b i c cells o f M. luteus. M e a s u r e m e n t s w e r e p e r f o r m e d as described in the legend to Fig. 10, at a cell c o n c e n t r a t i o n o f 12 g l - t an exciting f r e q u e n c y o f 30 Hz, a n d an exciting voltage ( z e r o - t o - p e a k ) o f + 0 . 7 V, save that K e N was p r e s e n t at t h e c o n c e n t r a t i o n s indicated.

of action of the inhibitor, b u t Js b e i n g forced towards static h e a d by its presence. In a similar vein, Fig. 11 shows a cyanide titration o f h a r m o n i c g e n e r a t i o n by aerobic, intact cells of this organism; low c o n c e n t r a t i o n s o f cyanide stimulate both second a n d third harmonics, which a r e subsequently d e c r e a s e d until at 20 m i cyanide they are no longer visible. T h e s e d a t a are exactly consistent with the k n o w n influence o f cyanide on the respiration of this o r g a n i s m (see above a n d ref. 22). F i g u r e 12 shows a similar titration of a n a e r o b i c cells, w h e r e t h e 3 r d h a r m o n i c is strongly inhibited d u r i n g the cyanide titration. I t m a y be m e n t i o n e d t h a t n e i t h e r cyanide n o r H Q N O h a d any m a r k e d effects u p o n the n o n l i n e a r dielectric p r o p e r t i e s o f the cells w h e n t h e f r e q u e n c y of excitation exceeded 100 Hz, a n d t h a t the H + A T P synthase inhibitor venturicidin [23] was also without effect at both low a n d high frequencies ( d a t a not shown).

0

E .~=

-lo -15

~o :-

-20

0

5

10

15

20

[KCNI/m, Fig. 12. E f f e c t o f c y a n i d e o n the n o n l i n e a r dielectric p r o p e r t i e s o f a n a e r o b i c cells o f M. /uteus. M e a s u r e m e n t s were p e r f o r m e d as d e s c r i b e d in th~ l e g e n d t o Fig. 10, a t a cell c o n c e n t r a t i o n o f 12 g I - t a n exciting f r e q u e n c y o f 30 I-Iz, a n d a n exciting v o l t a g e ( z e r o - t o - p e a k ) o f + 0.7 V, save t h a t K C N was p r e s e n t at the c o n c e n t r a t i o n s indicated.

432 20

0

="

-20 -30 1

10

100

1000

•

["rcquency/llz Fig. 13. Effect of frequency on the generation of harmonics by membranes vesicles of M. luteus. Measurements were performed as described in the legend to Fig. 7, except that membrane vesicles were present at a concentration of 50 g i-I, the voltage (zero-to-peak) across the outer electrodes was -!-0.7 V, and the frequency was varied as indicated. E a c h of t h e above e x p e r i m e n t s h a d i n d i c a t e d a clear r e l a t i o n s h i p b e t w e e n t h e type of h a r m o n i c s o b s e r v e d (odd vs. even) a n d t h e activity of t h e r e s p i r a t o r y chain. H o w e v e r , in i n t a c t cells r e s p i r a t i o n is e n d o g e n o u s , a n d t h e proximal e l e c t r o n d o n o r is u n c e r t a i n . It is t h e r e f o r e m u c h m o r e c o n v e n i e n t to study m e m b r a n e Vesicles. O s m o t i c a l l y - p r e p a r e d cytoplasmir, m e m b r a n e vesicles from r e s p i r a t o r y b a c t e r i a n o r m a l l y consist" of a mixture of so-called right-side-out, ~nside-out a n d " s c r a m b l e d " vesicles [24]; since N A D H is m e m b r a n e - i m p e r m e a n t , only inside-out, a n d to some e x t e n t s c r a m b l e d , vesicles c a n respire using e x o g e n o u s N A D H . T h e addition of N A D H (or an N A D H - g e n e r a t i n g system) to m e m b r a n e vesicles t h e r e fore provides" a c o n v e n i e n t m e c h a n i s m for t u r n i n g r e s p i r a t i o n on a n d off u n d e r o t h e r w i s e aerobic conditions. W e t h e r e f o r e p r e p a r e d m e m b r a n e vesicles f r o m M. l u t e u s cells a n d s t u d i e d t h e i r n o n l i n e a r dielectric properties. F i g u r e 13 shows t h e f r e q u e n c y - d e p e n d e n c e o f t h e ability of m e m b r a n e vesicles o f M . l u t e u s to g e n e r a t e harmonics. In a m a n n e r r a t h e r similar to t h a t observed in intact cells (Figs. 5, 7), two regions of f r e q u e n c y - d e p e n d e n c e m a y be observed, respectively below a n d above ca. 100 Hz. A~s expected, given t h e a b s e n c e o f a d d e d r e s p i r a t o r y substrate, t h e t h i r d hal~iionic is d o m i n a n t in t h e l o w - f r e q u e n c y region. T h e voltage w i n d o w for t h e low-frequency region is shown in Fig. 14, and, d e s p i t e t h e fact t h a t t h e vesicles a r e of course s m a l l e r t h a n t h e intact ceils from which t h e y a r e derived, is very similar to t h a t (Fig. 4) o f t h e i n t a c t cells (but contains, o f course, only a third h a r m o n i c , since e n d o g e n o u s r e s p i r a t o r y s u b s t r a t e s are now absent). Finally, to c o n f i r m t h a t t h e signal is i n d e e d d u e to t h e p r e s e n c e of t h e m e m b r a n e vesicles, Fig. 15 shows t h e c o n c e n t r a t i o n - d e p e n d e n c e of the. ability of m e m b r a n e vesicles to g e n e r a t e a third h a r m o n i c . T o c o n f i r m t h e d e p e n d e n c e of t h e type of h a r m o n i c on w h e t h e r or n o t t h e m e m b r a n e s w e r e p e r f o r m i n g e l e c t r o n t r a n s p o r t , we c o m p a r e d m e m b r a n e s in t h e absenc~ of respir~ltory s u b s t r a t e a n d those to w h i c h N A D H h a d b e e n a d d e d . F . i g u r e s 1 6 A ancl'16B show t h e i r respective n o n l i n e a r dielectric p r o p e r t i e s w h e n excited at 30 Hz. It m a y be observed (Fig. 16) t h a t t h e a d d i t i o n of N A D H causes

•

k

"

433

2 0

°

•

°

,

°

°

°

•

°

•

,

15 10 O

5 0

" 0

0.5 1 VoLfage ( z e r o - t o - p e a k ) / V

1.5

.

Fig. 14. Effect o f voltage o n the generation o f harmonics by m e m b r a n e vesicles o f M. iuteus. M e a s u r e m e n t s w e r e p e r f o r m e d as described in the legend to Fig. 13, except that m e m b r a n e vesicles w e r e present at a concentration o f 50 g t - I , the frequency o f excitation was 30 F h , and the voltage (zero-to-peak) across the o u t e r electrodes was varied as indicated.

an e n o r m o u s e n h a n c e m e n t of the 2nd and 4th harmonics at the e x p e n s e o f the 3rd harmonic (which had b e e n d o m i n a n t in the absence o f the respiratory electron donor). It may be m e n t i o n e d again that appropriate control experiments carried out in the absence o f m e m b r a n e s showed that neither oxidised nor reduced pyridine nucleotides, nor 0 2 , had any measurable effect in generating nonlinear dielectric responses. T h u s , these data provide the clearest possibl0 evidence that the generation o f e v e n - n u m b e r e d harmonics by M. luteus (in the low-frequency region) d e p e n d s u p o n t h e o n g o i n g activity o f the respiratory electron transport chain. Finally w e turned our attention to the high-frequency region ( > 100 H z ) . . A s a result o f the findings in intact cells o f M. luteus described above, w e again studied cells o f baker's yeast at the higher frequencie s. W e observed (data n o t s h o w n ) that cells o f baker's yeast w e r e also capable o f generating harmonics almost id.enti.cal to

2O • .

[

10

"

5 .,

0 0

20

40

60

[membranes]/g

80 t-I

•

.

.

100

Fig. I5. Effect o f vesicle concentration o n the genera2ion o f harmonics by m e m b r a n e vesicles o f M. luteus. M e a s u r e m e n t s w e r e p e r f o r m e d as described i n the legend to Fig. 14, with an exciting frequen~-y o f 30 Hz; an e x c i t i n g v o l t a g e (zero-to-peak) across the o u t e r electrodes o f + 0 . 8 V, a n d w i t h the m e m b r a n e vesicle concentration varied as indicated.

434

5

5

0 -5 -10 -15

-5

o

-20 -10 ~ 0

100

200

300

400

-25 0

500

!

100

t

|

200

I

[

300

a

400

•

F

500

Frequency/Hz

Frequency/Hz

Fig. 16. Effect of NADH on the generation of harmonics by membrane vesicles of M. luteus. Measurements were performed as described in the legend to Fig. 13, except that membrane vesicles were presenl at a concentration of 6 g I - t the frequency of excitation was 30 Hz, and the voltage (zero-to-peak) across the outer electrodes was + 0.8 V. (A) No NADH present. (B) 10 mM NADH was added 1 rain before measurement of the nonlinear dielectric spectra. t h o s e g e n e r a t e d b y M. luteus, w h e n t h e e x c i t i n g f r e q u e n c y w a s i n t h e r a n g e 0 . 6 - 2 kHz (the latter being the highest exciting frequency at which our equipment could conveniently measure), provided that the voltage (zero-to-peak) on the outer e l e c t r o d e s w a s in t h e r a n g e 5 : 0 . 5 - 0 . 9 V . T h i s l a t t e r e x p l a i n s w h y w e d i d n o t o b s e r v e t h e s e h a r m o n i c s a t h i g h f r e q u e n c i e s i n o u r e a r l i e r w o r k [1], w h i c h w a s carried out at higher field strengths, outside the voltage window. Since the h i g h - f r e q u e n c y n o n l i n e a r d i e l e c t r i c i t y d e p e n d e d o n t h e p r e s e n c e o f cells, b o t h f o r y e a s t a n d f o r M. luteus, w e t h e r e f o r e t u r n e d o u r a t t e n t i o n t o m e m b r a n e v e s i c l e s o f

M. luteus. F i g u r e 17 s h o w s t h e g e n e r a t i o n o f n o n l i n e a r d i e l e c t r i c i t y b y m e m b r a n e v e s i c l e s o f M. luteus e x c i t e d a t 1 k H z . T h e v o l t a g e w i n d o w ( F i g . 18) is s i m i l a r t o , t h o u g h •

~

-10

o

=15

•

i

•

i

•

g

a

•

i

•

i

-20 -25

'

0

2

4

. . . . .

6

8

''

10

°*'

12

14

ttarmonie number (Frequency/kHz) Fig. 17. The generation of nonlinear dieleetrieity by membrane vesicles of M. luteus when excited at 1 kHz. Measurements were performed as described in the legend to Fig. 14, except that membrane vesicles were present at a concentration of 50 g I-X,-the frequency of excitation w,~ 1 kHz, and the voltage (zero-to-peak) across the outer electrodes was =l=0.7 V.

435 2O

~.

.-

10

E

0

-~.=_

-10

~"

-30 0

0.5 1 1.5 Vo[fage ( z e r o - t o - p e a k ) / V

2

Fig. 18. Effect o f v o l t a g e o n the g e n e r a t i o n o f h a r m o n i c s by m e m b r a n e vesicles o f M. luteus w h e n excited at high frequencies. M e a s u r e m e n t s w e r e p e r f o r m e d as d e s c r i b e d in t h e l e g e n d to Fig. 17, except that m e m b r a n e vesicles w e r e p r e s e n t at a c o n c e n t r a t i o n o f 25 g I - ~, t h e f r e q u e n c y o f excitation was 1 kHz, a n d t h e v o l t a g e (zero=to-peak) across the o u t e r e l e c t r o d e s w a s v a r i e d as indicated.

slightly h i g h e r t h a n , t h a t o f t h e i n t a c t cells (Fig. 4). T h e c o n c e n t r a t i o n - d e p e n d e n c e of t h e h a , , , o n i c s ge'aerated by m e m b r a n e vesicles w h e n excited at 1 kI-Iz is shown in Fig. 19, w h e r e it may be observed t h a t it is b o t h m o n o t o n i c a n d unselective towards p a r t i c u l a r ha,,c, onics. In this f r e q u e n c y r a n g e (above some 100-200 I-Iz), as in intact cells, t h e ha~,,ionic p a t t e r n s were practically i n d e p e n d e n t o f t h e p r e s e n c e o f N A D H ( a n d r e s p i r a t o r y or H+oATP synthase inhibitors; d a t a n o t shown), i.e. o f o n g o i n g r e s p i r a t o r y or A T P synthetic activity. T h u s , since t h e esicles c o n t a i n only p r o t e i n s a n d lipids, o n e is led to implicate t h e lipid p o r t i o n o f t h e vesicle m e m b r a n e s as t h e source o f t h e n o n l i n e a r dielectricity in t h e Eigho f r e q u e n c y range. It t h e r e f o r e s e e m e d p r u d e n t to c h e c k w h e t h e r p u r e p h o s p h o l i p i d liposomes, lacking p r o t e i n , m i g h t also be able to g e n e r a t e h a r m o n i c s w h e n excited above 100 Hz.

10

"E

0

o

r. '-o-

. ~

-10 -20

o

-30 1

10 [membra'nes]/g

100 [-I

Fig. 19. E f f e c t o f vesicle c o n c e n t r a t i o n o n t h e g e n e r a t i o n o f h a r m o n i c s b y m e m b r a n e vesicles o f 3,f. luteus. M e a s u r e m e n t s w e r e p e r f o r m e d as d e s c r i b e d in t h e l e g e n d to Fig. 17, with a n exciting frequenc~ o f 1 k H z , an exciting v o l t a g e (zero=to=peak) across t h e o u t e r e l e c t r o d e s o f 4-0.8 V, a n d with the m e m b r a n e vesicle c o n c e n t r a t i o n indicated.

.

.

.

436 15

s

-10 -15 0

500

1000

1500

2000

i"requcncy /llz Fig. 20J Effect of frequency on the generation of harmonics by liposomes. Liposames were prepared as described in the Experimental. Nonlinear dielectric spectra were measured as described in the legend to Fig. 18. ThE voltage (zero-to-peak) across the outer electrodeswas d:7 V and the frequency was varied as indicated. F i g u r e 20 shows t h e f r e q u e n c y - d e p e n d e n c e o f t h e ability of liposomes to g e n e r a t e h a r m o n i c s , w h e r e it may be observed t h a t d a t a similar to those observable in m e m b r a n e s (i.e. the g e n e r a t i o n o f a p l e t h o r a of b o t h odd- and e v e n - n u m b e r e d h a r m o n i c s ) are o b t a i n e d , a l t h o u g h in t h e s e very small liposomes significant h a r m o n i c s a p p e a r only w h e n t h e exciting voltage (zero-to-peak o n t h e o u t e r electrodes) exceeds 4 V (not shown). T h u s , it would a p p e a r t h a t it is t h e lipid p o r t i o n of t h e cytoplasmic m e m b r a n e of M. luteus ( a n d o t h e r organisms) w h i c h is p r e d o m i n a n t l y responsible for the g e n e r a t i o n of h a r m o n i c s w h e n t h e cells are excited at f r e q u e n c i e s above 100-200 Hz.

DISCUSSION W e have previously described in detail t h e c o n s t r u c t i o n o f a dual-cell, fourterminal, n o n l i n e a r dielectric s p e c t r o m e t e r , a n d have d e m o n s t r a t e d t h e g e n e r a t i o n of n o n l i n e a r dielectricity by y e a s t cell s u s p e n s i o n s [1-3]. T h i s was m a n i f e s t as haffftonics w h e n cells were excited, w i t h i n a p p r o p r i a t e voltage a n d f r e q u e n c y windows, by sinusoidally m o d u l a t e d electric fields of a single frequency. I n this system, i n h i b i t o r studies revealed t h a t t h e m a j o r source of t h e n o n l i n e a r dielectricity observed was t h e H + - A T P a s e p r e s e n t in t h e p l a s m a m e m b r a n e o f this o r g a n i s m [1-3]. T h e type of h a r m o n i c s observed d e p e n d e d u p o n w h e t h e r t h e cells w e r e resting or glycolysing, t h e f o r m e r case causing the g e n e r a t i o n of o d d - n u m b e r e d h a r m o n i c s whilst ceils w h i c h w e r e actually m e t a b o l i s i n g glucose a n d g e n e r a t i n g A T P via substrate-level p h o s p h o r y l a t i o n g e n e r a t e d e v e n - n u m b e r e d h a , u o n i e s [1]. T h e s e findings could be explained on t h e basis of t h e ability o f electric fields to alter t h e c o n f o r m a t i o n a l dynamics of ( e s p e c i a l l y ) m e m b r a n e p r o t e i n s [26-43], whilst t h e c h a n g e from odd- to e v e n - n u m b e r e d h a r m o n i e s could be a c c o u n t e d for in t e r m s o f a simple double-potential-well m o d e l [2].

437

HO.NO NADH --,,.. Fp--.~ M Q

. b-~

.\

low CN-

o.((:1,3)~--~02 o ~high CN02

Fig. 21. Simplified diagram of the branched respiratory chain of Micrococcus luteus, and of the sites of inhibition of electron transport by cyanide and H Q N O [22,46]. The probable involvement of differing redox states o f the menaquinone moieties in a so-called Q-cycle is not shown. MQ, menaquinone; b, cytochrome(s) b; o, cytochrome o; a, cytochrome a; Fp, flavoproteins.

In the p r e s e n t work, we have used this n o n l i n e a r dielectric s p e c t r o m e t e r system to study t h e g e n e r a t i o n o f n o n l i n e a r dielectricity by the obligately aerobic, heterotrophic, respiratory b a c t e r i u m Micrococcus luteus. W e showed, for the first time in a r e s p i r a t o r y system, t h a t intact cells of M. luteus g e n e r a t e d ha~'~onics w h e n excited with single sinusoids (Figs. 1 , 5 ) . T w o f r e q u e n c y windows w e r e observed, respectively below ( " l o w f r e q u e n c i e s " ) a n d above ( " h i g h f r e q u e n c i e s " ) ca. 100 H z (Figs. 5, 7). T h e low-frequency region showed a voltage window o f some 5 : 0 . 4 - 1 . 6 V (0.5-2.1 V c m -~, zero-to-peak, as j u d g e d on t h e o u t e r electrodes, 3 0 - 4 0 0 m V c m - t as j u d g e d on the i n n e r electrodes). In the low-frequency region, a n a e r o b i c (non-respiring) cells g e n e r a t e d o d d - n u m b e r e d harmonics, whilst aerobic (respiring) cells g e n e r a t e d e v e n - n u m b e r e d harmonics (Fig. 3). Figure 21 shows a simple r e p r e s e n t a t i o n o f t h e b r a n c h e d electron t r a n s p o r t chain of M. luteus [21], indicating t h e site of action o f a p p r o p r i a t e respiratory inhibitors. In t h e low-frequency region, we f o u n d t h a t the inhibition o f respiration by H Q N O (Fig. 10) or by" cyanide (Fig. 11) caused the a p p e a r a n c e of o d d - n u m b e r e d h a , m o n i e s in aerobic cell suspensions. H i g h e r concentrations o f cyanide (but not o f H Q N O ) cause t h e d i s a p p e a r a n c e o f all harmonics, in both aerobic (Fig. 11) a n d a n a e r o b i c cells (Fig. 12). T h e s e facts a r e consistent with t h e known effects o f these inhibitors o n electron t r a n s p o r t via t h e b r a n c h e d respiratory chain o f this o r g a n i s m [21,22], a n d suggest t h a t the m a j o r source o f n o n l i n e a r dielectricity within the r e s p i r a t o r y chain is either in the b r a n c h to eytochrome bss8 o r proximal to t h e site at which H Q N O binds (Fig. 21). Since intact ceils of M. luteus could respire e n d o g e n o u s l y , a n d in o r d e r to excluo¢ cytoplasmic processes, it was o f interest to study t h e effects of respiration on t h e g e n e r a t i o n of n o n l i n e a r dielectricity by this o r g a n i s m a t the m e m b r a n e vesicle level. W e f o u n d t h a t m e m b r a n e vesicles of M. luteus g e n e r a t e d h a r m o n i c s with p a t t e r n s (voltage a n d frequency windows) similar to those f o u n d in intact cells (Figs. 13-19). In the absence o f a n electron donor, t h e third h a r m o n i c was d o m i n a n t (Figs. 13-16), as expected. However, in t h e low-frequency region, the

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a d d i t i o n of N A D H to t h e m e m b r a n e vesicles caused the r e p l a c e m e n t o f the o d d - n u m b e r e d harmon:~cs by substantial e v e n - n u m b e r e d h a r m o n i c s (Fig. 16), providing extremely clear-cut evidence t h a t the type of harmonics observed d e p e n d e d strongly on w h e t h e r or not the cells were carrying out respiratory electron transport. It is evident t h a t this p h e n o m e n o n might be exploited for t h e m e a s u r e m e n t o f respiratory activity in situ. In t h e high-frequency range, intact cells of M. luteus (and also o f Saccharomyces cerevisiae) g e n e r a t e d b o t h odd- and e v e n - n u m b e r e d h a r m o n i c s (Figs. 6 - 9 ) , w h e t h e r the cells were aerobic or anaerobic. Similarly, w h e n excited in t h e high-frequency region, b o t h odd- a n d e v e n - n u m b e r e d h a r m o n i c s were g e n e r a t e d by m e m b r a n e vesicles, w h e t h e r or not the m e m b r a n e s were carrying out electron t r a n s p o r t (Figs. 13, 17-19). W e also f o u n d t h a t liposomes consisting o f pure phospholipids showed very similar p a t t e r n s o f h a r m o n i c s (Fig. 20), strongly suggesting that the high-frequency n o n l i n e a r dielectricity may be ascribed to the dynamic p r o p e r t i e s o f the lipid p o r t i o n o f the m e m b r a n e s of cells and vesicles (and see also refs. 44,45). It is concluded, on the basis o f t h e p r e s e n t work, a n d o f that described previously [1-3], that b o t h respiratory a n d f e r m e n t a t i v e microorganisms can generate n o n l i n e a r dielectricity w h e n excited within the a p p r o p r i a t e voltage and frequency windows, and t h a t this n o n l i n e a r dielectricity is caused, in these circumstances overwhelmingly, by the electric field-induced c o n f o u n a t i o n a i dynamics of t h e i r membrane-located enzymes. F u t u r e work will be directed towards a m o r e m o l e c u l a r characterisation of these effects and to the more rapid acquisition o f n o n l i n e a r dielectric spectra. ACKNOWLEDGEMENTS

W e are i n d e b t e d to the Wolfson F o u n d a t i o n for financ:.al s u p p o r t o f this work. W e also wish to t h a n k Dr. Axseny Kaprelyants for advice on t h e growth a n d p r o p e r t i e s of M. luteus. REFERENCES 1 A . M . Woodward and D.B. Kell, Bioelectrochem. Bioenerg., 24 (1990) 83. 2 A.M. Woodward and D.B. Kell, Bioelectrochem. Bioenerg., 25 (1991) 395. 3 D.B. Kell and A.M. Woodward, in H.V. Westerhoff (Ed.), Biothermokinetics, Inter~-ept Press, London, in the press. 4 H.P. Schwan, Adv. Biol. Med. Phys. 5 (1957) 147. 5 E.H. Grant, G.P. South and R J . Sheppard, Dielectric Properties o f Biological Molecules in Solution, Oxford University Press, Oxford, 1978. 6 0 . Schanne and E.R.P. Ceretti, Impedance Measurements in Biological Cells, John Wiley, Chichester, 1978. 7 R. Pethig, Dielectric and Electronic Properties of Biological Materials, John Wiley, Chichester, 1979. 8 D.B. Kell and C.M. Harris, J. Bioelectricity0 4 (1985) 317.

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