Adaptation of Bacillus FTU and Escherichia coli to alkaline conditions: the Na+-motive respiration

Biochi.+~ca et Biophv+i< "a +~l<+t+¢, I ()98 ¢ I o91 ) q 5 - 1 0 4 .~" It, D1 [-Isevicr Scicnct: P u b l i s h e r s B.V. All right,; r e s e r v e d 01)[15-272~/~1/$()3.51]

t) 5

B B A B I O 43521

Adaptation of Bacillus FTU and Escherichia coli to alkaline conditions: the Na+-motive respiration A r m i n e V. Avetisyan, Pavel A. Dibrov+ A n n a L. Scmeykina, V l a d i m i r P. Skulachev a n d M a x i m V. Sokolov ,4..'¢. Fh'lozersky b~ritz~t~, ,ff l't~y~t('o+(Twmicai t¢iology, .'1,fo,,+'ov~. Stat¢' ('~zit ¢'t;~ttx, .~l+,,,c.,,,.v ~1 r..~.k%"~ j ( R e c e i v e d 24 Ap+il It~91 ) (Rc~i,,cd manu~,cript r c c c i ~ c d 21 ,~,tlgtlM FJgl )

Key ~ortl:,: S o d i u m r c , , p i r a l o l y c h a i n : A l k a l i n e a d a p t a t h ~ n : N A l ) l l - q u i n o n ~ ~cd,acta~,c, Na +: I)xida,,*:. Na +: ~ t?acflh+~ 1- 17;. I:+ ~oli )

M e c h a n i s m s of Na + t r a n s p o r t into the inside-out s u b c e l l u l a r vesicles of alkalo- a n d h a l o t o l e r a n t Bacillus F T U a n d of E s c h e r i c h i a coli grown at different pH have been studied. Both m i c r o o r g a n i s m s growing at pH 7.5 are shown to possess a system of the r e s p i r a t i o n - d e p e n d e n t Na + t r a n s p o r t which (i) is i n h i b i t e d by p r ~ t o n o p h o r o u s uncoupler, by , ~ p H - d i s c h a r g i n g a g e n t d i e t h y l a m m o n i u m IDEA) acetate, by m i c r o m o l a r cyanide a r r e s t i n g the H +-motive respiratory c h a i n , a n d by a m i l o r i d e , a n d (ii) is resistant to the N a + / H + a n t i p o r t e r m o n e n s i n a n d to Ag +, i n h i b i t o r of the N a + - m o t i v e r e s p i r a t o r y chain. Growth at pH 8.6 strongly changes the activator a n d i n h i b i t o r p a t t e r n . Now (I) p r o t o n o p h o r e s t i m u l a t e s the Na ÷ t r a n s p o r t , 12) DEA acetate is without effect in the absence of p r o t o n o p h o r e a n d is s t i m u l a t i n g in its presence, !3) a m i l o r i d e a n d low cyanide are ineffective, (4} m o n e n s i n a n d Ag + completely a r r e s t the Na + a c c u m u l a t i o n in the vesicles. I n d e p e n d e n t of pH of the growlh m e d i u m , (a} v a l i n o m y c i n is s t i m u l a t o r y for the Na + t r a n s p o r t , (b) Na + ionophore ETH 157 is inhibitory and, (ck Na + t r a n s p o r t c a n be s u p p o r t e d by NADH -+ f u m a r a t e as well as by ascorbate ITMPDJ --, O z electron transfers. Growth at a l k a l i n e pH results in the a p p e a r a n c e of ascorbate I T M P D ) oxidation resistant to low a n d sensitive to high cyanide c o n c e n t r a t i o n s . These r e l a t i o n s h i p s are in a g r e e m e n t with the concept
! nt roduct ion Recent progress in bioenergetic studies has resulted in the discovery of the Na+ cycle which can effectively substitute fl)r the tt+ cycle in certain bacteria! species (for reviews, scc Refs. I - h ) . A d a p t a t i o n to a;kalint

A b b r e v i a t i o n s : d ~ l ~ , a n d . . 1 ~ , , + . t r a n s m c m b r a n c difference~, in e l e c t r o c h c r n i c a l [1: a n d Na ~ potential,,, respectively: .l~h. tran,,+ m e m b r a n e eleclri¢ p o l e n l i a l diflk:rcnce; ( ' ( ' t + P , m-chh+rt~carbonylc y a n i d e p h c n y l h y d r a z o n c ; I)['-'A, dicth31amin¢; [-TII 157, N . N ' - d i b e n z y I - N . N ' - d i p h e n y l - l . 2 - p h e n y l e n e d i a c c t a m i d c : l I O N ( ' ) . 2+hcptyl4-hydroxyquin~dinc N-oxide: TMPI). N.N.N'.N'-lclranlethyl-pphenylenc diamine: TPT. triphenyllin. C o r r e s p o n d e n c e : V.P. S k u l a c h c v , A . N . 13elozcrsky In'*titul¢ *~t P h y s i c o - C h e m i c a l [:liolot.~, M o s c o w 3~l~.llc t l n i v c r s i l y , L c n g o ~ , M o s c o w 1 It.~gqtL U.S.S.R.

c o n d i t i o n s seems an obvious reason for such a substitution. L o w 1t ~ c o n c e n t r a t i o n in tile mediutn creates kinetic difficulties for all the systems dealing w i t h H+ ion~. ILven a more i m p o r t a n t t h e r m o d i n a m i c p r o b l e m arises when [H ' ] outside the cell appears to be lower than [H +] inside. In this case, electric p o t e n t i a l (..1~), the cell i n t e r i o r negative, is c o u n t e r b a l a n c e d by o p p o site .1ptt. As a result, H + ions p u m p e d from the cell man hardly p e r f o r m w o r k when coming back to the cytoplasm as long as they move from the c o m p a r t m e n t of low [H ~ ] tt~ that of high [..'4 * ]. T o overcome these difficulties, one may postulate a strong increase in Adh a n d / o r in the n u m b e r of H+ ions transported into the cell per, e.g., o n e A T P molecule tbrmcd by H +-ATP synthase+ Substitution of N a ' for H+ seems a more radical solution of the prt,blem. Such a possibility was first

96

m e n t i o n e d ten years ago by Krolwich and her colleagues [7]. In ~b14 we put forward the concept that adaptation to alkaline c, mdi!it~n~ is :lcc~m~panied bv induction of the primary, Na" pumps g e n e r a t i n g A~N: ,. by Na ~ extrusion from the cell [/4]. In the present paper, this suggestion was confirmed by e x p e r i m e n t s on Bac. F T U and E. co//. It was fl~und that growth u n d e r alkaline conditions results in the a p p e a r a n c e of the N a ' motive N A D H - q u i n o n e rcduclasc and terminal oxidasc a~ tivities. T h e same bacteria growing at neutral p i t were shown to possess only the H +-motive respiratory chain. They extruded Na" in a A ~ u .-driven fashion, the process being mediated by the amiloridesensitive N a + / H ' ant{porter. T h e ant{porter proved unnccessaD' trader alkaline conditions when the primary Na " pump,,, were operative. Materials and Methods

Alkalo- and hatomlerant Bac. i,'TU was isolated in our laboralory itS,Ill]. The growth m e d i u m c o n t a i n e d lt.5 M Na('l, I11 mM K('I, 15 mM ( N H ~ ) 2 S O 4. 2 mM K H , P O a , 5 mM MgSO 4, I × 111 5 M FcSO 4, 0.1 mM E D T A , 20 mM Tris-H('i, pH 8.6 or 7.5, a n d 61) mM sodium succinate as the only energy a n d carbon source. Bacteria ~,cre grown aerobically at 37 ° C a n d constant pH. Inside-out subccllular vesicles wcrc obt~dned from Bac.FI'U cells by means of ~onication an described elsewhere [10]. E. coil strain KI2 D o c - S ( l a c i z + y ' a * . p r o , lrp . his , met ) was ,~ent by Professol L. Grin{us. Cells wcrc grown aerobically at 3 7 ° C up to the middle exponential phase in 22 mM K t t , P O ~ , 211 mM N a , H P O 4, h ) m M NaCI, I mM MgSO 4, 50 m M sodium glycylglycine, p l i 8.6 or pH 7.5, with the addition of marker a m i n o acids, i.e., proline, tryplophan, histidinc and moth{on{no 1t1.11/4 mg ml i), 0.(15£/ yeast extract and {~0 mM sodium succinalc. The growth rate at p l l 8.6 comprised t~{)c; of that at p H 7.ft. "1"o obtain the i'J. ctdi inside-out subccllular vesicles, the cells were s c d i m c n t e d at 751111x g, il1 rain, washed with 1511 mM NaCI. 5 m M MgSO 4, 10 mM Trio{no, pH 8.2 or t t E P E S , pH 7.5, and incubated at 3 3 ° C liar 30 rain in 11.2 M sucrose, 5 mM MgSO 4 and 10 mM Tricinc. pH /4.2. After incubatioa, the cells wcrc scdim e n t e d once more u n d e r the same conditions, susp e n d e d in 0.1 M K : S O 4, 311 mM MgSO 4, l0 mM Tricinc, ptt 8.2, or |lopes, ptt 7.5, and lysozymc 111.3 mg x ml t) and i n c u b a t e d for 45 rain at 3 7 " C . The resulting spheroplasts were centrifuged at 9800 × g for 10 rain. ]'he s e d i m e n t was suspended in 0.1 M K_,SO 4. 5 mM N a z S O 4, 5 mM MgSO a, 10 mM Tricine, p|-I 8.2, or 10 mM Hepcs, pH 7.5, serum a l b u m i n {I mg ml t), 4 mM dithiothrcitol and 11.5 mM phenylmethylsulfonyl fluoride. The suspension was treated with F r c n c h press (I(XX) Ib inch-'). The cell debris was removed by ccnlrif-

ugation at 1 4 0 0 0 × g for 15 min. F r o m the supern a t a n t , the subcellular vesicles were s e d i m e n t e d at 4/451~J x g for l h. Tile vesicles were s u s p e n d e d in 'L3-0.4 ml of 0.1 M K 2 S O 4, 5 m M N a 2 S O 4, 5 m M MgSO 4, 20% glycerol, l0 m M T r i c i n e , p H 8.2, o r Hepes. p H 7.5, a n d stored at the liquid n i t r o g e n t e m perature. in the majority of the experiments, the Na + u p t a k e by the subcellular vesicles was m e a s u r e d using gelfiltration and the c e n t r i f u g a t i o n p r o c e d u r e after P e n e f . sky [111. T h e process of the r e s p i r a t i o n - d e p e n d e n t N a + u p t a k e was initiated by a d d i n g T M P D o r N A D H . T o stop the reaction. 11.115 ml of the i n c u b a t i o n mixture was centrifuged at/4110 × g for 1.5 rain in a 21 m m long gel-filtration c o l u m n ( D = I0 ram) filled with S e p h a d e x G-511 coarse p r c - c q u i l i b r a t e d with buffer solution (0.1 M Tris-H_,SO 4, pH 8.2, a n d 10 m M MgSO4). T h e buffer c o n t a i n e d ( 1 - 2 ) . i11 ~' M N a -~. T h e e l u a t e was diluted ten-fold with bid{stilled water a n d [Na +] w a s m e a s u r e d with a PFM flame p h o t o m e t e r . T h e S e p h a d e x filtration p r o c e d u r e was shown to decrease the extravcsicular [Na ' ] by factor 5 - 1113. S t a n d a r d deviation of the m e a s u r e m e n t was f o u n d to be a b o u t 7%. In some experiments, the Na + uptake was m o n i tored using ~-'Na ~. For this purpose, s u b c e l l u l a r vesicles were i n c u b a t e d at 2 5 ° C in buffer A (0.1 M K 2 S O 4, 2.5 m M Na , S O 4. I11 m M MgSO 4, 50 m M T r i c i n e - K O H , p|"l 8.2), s u p p l e m c : l t e d with 50 m M D E A acetate, 15 mM potassium ascorbate a n d Z?NaCI (4 ,u,Ci m l - * ) . Respiration was initiated by a d d i n g 0.5 m M T M P D . T o arrest the process, 0.1 ml samples were withdrawn, put into I ml cold buffer A a n d passed through a nitrocellulose Vladipore filter (pore size, 1t.9 ~tm). T h e n , the filter was washed three times with 5 ml cold buffer A and dried; the radioactivity was m e a s u r e d b~ liquid scintillation counting. T h e results o b t a i n e d by this method proved to be cssentmlly the same as by the I]ame p h o t o m e t e r techniques. T o measure the volume of vesicles, a p e n e t r a t i n g spin-label, 2,2'.6,6'-tetramethyl-4-ox~piperidine l-oxyl I T E M P O , i • 10 5 M) was used [131. T h e E S R signal was measured with an RE-1307 ESR spectrometer. T h e volumc of the B a c . F T U or E. coil vesicles was fotmd to be about 11.118 or 0.07 /al x mg p r o t e i n - i rc~,pcctively, ()xygen c o n s u m p t i o n was m e a s u r e d wir_h a s t a n d a r d oxygen Clark-type electrode. N A D H - f u m a r a t e reductase activity was m o n i t o r e d in the p r e s e n c e of high cyanide c o n c e n t r a t i o n s using an A m i n c o spcctrophotometer. Results

Set o f spectfic actit ators and inhihitors affecting two mechanism~ o f Na ' tran.~port O u r previous studie~ on Bac. F T U cells a n d subeeilular vesicles have shown that these bacilli growing at

~7 TABLE I The aclit'ation a n d inhibiticm ptHlerlt,~ ]~r two mecl:uld.~m.s o f the ,N'a " uptake hy the m~-ide-ottt bacterial tesick',: Activators

and inhibitors

Effect on the Na + u p l a k c H' pump and Na ~ / H ' a n t i p o r t e r

Na'

CCCP

inhibition (,..l,~lt • decrease)

stimulation (AW --~ApNa. Jpli transition)

DEA acetate

inhibition (ApH decrease)

no effect

CCCP + DEA acetate

inhibition (A~. H • d e c r e a s e )

Valinomycin

stimulation ( J ~ -, hpll transition)

maximal ',,tim uia t|on (steady ...1~ d e c r e a s e )

inhibition

no effect

Amitoride

pump

m a x i m a l stimulation

(AW decrease with m~ Aplt tt,rmation)

(of N a ~ / I t " antiporter)

effect

Monensin

no

Low cyanide concentration

inhibition ( o f t l ~-motivc

(K i 2X l 0 -f' M)

terminal oxidase)

Ag

no effect

inhibition (modification of Na 'motive N A D I t - O rcductasc, resulting in Na ' c o n d u c t a n c e )

ETH 157

inhibi0on (Na" conductance)

inbibition (Na" conductance)

inhibition ( . . l p N a --, ..It/, transition) no effect

alkaiine pH possess two types of respiration-linked energy transducers: namely, N a - and H*-motive respiratory chains. Both chains include at least two coupling sites, i.e. NADl--l-quinone reductase and terminal oxidase [1(1,12]. in such a complicated system, Na" transport may, in principle, bc mediated alternatively by the primary Na* p u m p or by cooperation of the H +-motive respiration and the Na + / H - ' - a n t i p o r t c r . T o discriminate b e t w e e n these possibilities, it seems reasonable to use inside-out subcellular vesicles rather than such a complicated subject as intact cells, in the vesicles, alternative mechanisms of the Na + uptake can easily be distinguished by m e a n s of specific ihhibitors and activators listed in Table !. The most dramatic difference between responses of the H + p u m p + N a ÷ / H ~ antiporter and Na + p u m p mechanisms is revealed when a p r o t o n o p h o r e uncoupler, e.g., C C C P is added. This agent must completely block the Na ~ uptake m e d i a t e d by the former system due to A~,tt- dissipation. As to the latter one, CCCP

must stimulate the N a uptake, discharging the N a p u m p - p r o d u c e d J W and thereby allowing the largescale Na ~ transpor! ( , 3 ~ t - - , A p N a transition). In this case. A p H will also arise as a c o n s e q u e n c e of the electrophorctic, C C C P - m c d i a t e d H ' cfflux. ApH discharge by a salt of p e n e t r a t i n g weak base and weak acid, e.g., D E A acetate, will increase the efficiency of CCCP in discharging .3qr, generated by thc Na ~ pump. in the absence of CCCP, D E A acetate will be without effect. In the case of thc A ~ t t . - l i n k e d mechanism, D E A acetate must be inhibitory due to the dissipation of ApH, the driving force of the Na-~/H + antiportermediated Na + uptake. Valinomycin ( + K " ) m u s t effectively substitute for CCCP + D E A acetate in the case of the Na ~ pump. It will also be stimulating for the alternative mechanism due to the Aqt --, 'tpH transition. Amiloride, the N a ~ / t t * antiporter inl:Alzitor must be (i) inhibitory when the process is ..l~l,.-linked cr (it) without effect when the Na ~ pump is inw)lvcd provided that the Na* p u m p is resistant to this compound. O n the other hand, artificial N a * / H + antiporter m o n e n s i n must inhibit the Na" p u m p - m e d i a ted Na* uptake because of the ApNa --,.3pH conversion. it seems to bc without effect in the case of the H + p u m p if the Na + / H ~ antiporter is (a) clcctroncutrai and (b) sufficiently active to maintain the maximal rate of the Na " transport. As it was shown in our group [t2], the H '--motive terminal oxidase of Bac.FTU is sensitive to micromolar cyanide while with the Na '-motive one requires much higher cyanide c o n c e n t r a t i o n to be blocked. Thus the H + r e s p i r a t i o n - d e p e n d e n t N a ' uptake should be scnsitive to low cyanide c o n c e n t r a t i o n which should bc without effect upon the N a uptake d e p e n d e n t on the N a ' respiration. Low Ag" concentrations which block the N i t ' - m o tive N A D H - q u i n o n e rcductasc [14] inducing th~,reby the Na ~ conductance [15]. must specifically inhibit the Na ~ uptake supported by the Nit~-respiration activity. As to artificial Na ~ ionophorc E T H t57, it must prevent formation of the Na ' gradient created either by Na '-respiration or by H +-respiration. The inhibiting effect of E T H 157 indicates that Na" is transported uphill (for the downhill Na" transport, ETH 157 must be stimulatory). Growth at p H Z5: cooperation o]" the H '-motive re.wiration and Na */ t l "~ atttiporter Fig. I shows the respiration-supported transport of Na * into inside-ou! subcellular vesicles from Bar'. FTU grown at pH 7.5. Respiration was initiated by adding N A D H (A, B) or T M P D (C). In thc former case, fumarate and I mM KCN were present as an electron aceeptor a n d a terminal oxidase inhibitor, respectively. In the latter case, the m e d i u m contained ascorbate

tJl,l instead of fumaratc und cyat]idc. In ;ill the sample;4, 4 × I() " M T P T ~ a s added to increase the m a g n i t u d e of the N a ' uptake . (The T P T tsflccl xtas most probably duc to the inhibition o f l h e Na+ c o n d u c t a n c e via the F. parts of N a ' - A ' l ' P a s c .!','privcd of faclor Fi; N.N'-dicyclohcxyl carbodiimidc was found to substilutc for TP-I" [12,15]). It is sccn th,'. the ctcctron transfer in both initial ( N A D H -+-+fun,aratc) and terminal (ascorbaic ~ O~) spans of ,he respiratory chain could suppor~ the Na uptake (Fig IA, B and C, respectively). The Na ~ tra-lsport was strongly stimulated by valinomycin a n d inhibited by the p r o t o n o p h o r o u s u n c o u p l e r , CCCI ~, and the N a ~ / H " a n t i p o r t c r inhibilor, amiloridc (Fig. IA, C). The process was resistant to A g ' (Fig. IA) and to the artificial N a ' / H ' a n t i p o r t c r m o n c n s i n ;is well (Fig. IB). The salt of p e n e t r a t i n g ~ c a k base ( D E A ) and wcck acid (acetate) was sho~vn to decrease the N;.I"

uptake

(Fig.

I l l ) . I_or, c y ; t n i d c

Stlongly

inhibited

the Na + iranspt+rt (scc bchlw, Fig, 41:1).

The N a ' uplakc was completely abolishcd by Na ' i o n o p h o r c E T H 157 (Fig. 1C), indicating that the uphill Na" transport takes place. This conclusion is in agrecm c n t with the calculation of internal [Na +] after the Na + uptake which proved to reach 250 mM (the Na + c o n c e n t r a t i o n in lhc i n c u b a t i o n m e d i u m was 5 raM). i n the calculation, we look into account the intravesicular volume which was estimated as described in Materials a n d Methods. Similar rcsponscs wcrc observed when the vesicles from E. coil grown at pH 7.5 were used. A c o m p l e t e respiratory chain ( N A D H - - - > O 2) was studied. H e r e also valinomycin stimulated a n d C C C P abolished the Na* uptake (Fig. 2A). Both amiloride and D E A acetate wcrc inhibitory (Fig. 2B). Again. the c a l c u l a t i o n p o i n t e d to the uphill Na + transport. All these relationships c o r r e s p o n d exactly t o those expected a s s u m i n g thai the Na * transport, carried out by the Na * / H ~ a n t i p o r t c r utilizing ,.IpFl, is formed by the H '-motive respiratory chain (scc above, T a b l e I). b ~t" valirlo

/

14 A

e./"

"~ 12

//~

p

,0. •

/,~/'

o

i

,

-~ 0i

/.t/" ~

//

,~

/ .21

~

v a l i n o . Ag ÷

o ."

10

/ /

,

if

~j---

//

+o S~"X----~-t::-;Z:-~i . . . . . . . . . == I 0

O

i0

20

30

6'

• ~ ' A - valino. monensin

e//~i///••

=n

i

valino. BEA a c e t a t e

? /

/"

no additions

~.zcccP .-

volino

2,

amiloride

40

0 0c

r~o Time< s

lo

;to

So

40

S~0 tim-e , "

20" c

oo

cz

/I

,::: 10

/

no ,e,.ddqt'ons

o

CCCp

-~ ~

t... :.--~, 'F--%: "'-.. "ol'r'°.°~'il°"d~

~-

vohho. ETH 06

'0

~0

30

40

50 Time(s[

Fig. I. N u uptake h) Ihc insith.'-t~ul subccllul,ir vesicles tram Bac. F T U grown at p t l 7.5. The N a ' tiansporI "~,'m, s u p p o r l c d by N A D I I -+ furmtrat¢ (,'%. i:~) or a,,,~:(~rb~dc -~ O - ( ( ' ) elL'titan Iranslcr~,. Incubation m i x l u r c , (I.I M K : S ( ) 4, 2,5 m M N a 2 S O 4. 10 m M M g S O 4, 50 rnM T r i c i n c - K ( ) l l . p l l ~.2, 4- Ill " M TP'I'. vesicle,,, h.2 (A, B) and 4.7 (('1 mg p r o l c i n ml I. In A and H, lhc mixture was supph:mented w i l h 5 m M tl*~ta-,siunl tumarate altd I m M K(,'N, In (,', 15 m M pota~,sium ascorhate v, at, prcscnl. A t zero lime. 2 m M N A D l t (A. B) or 1).5 m M T M P D ( ( 3 v~crc ;iddL'd h) initi~Jl¢ ~lcL'tl~n trunsfer. ()thL~ additions. 1 ' I0 ' M valinomycin, I - I0 : M A g N O , . I - 10 ~ M C('C'P, 2. l0 "j M ( A ) or 5" 10 4

M ( ( ) a m i l o f i d c , 50 m M D E A acetate, I • l(I s M moncm, in. x. I() ~' M E T [ ! 157.

Growth at p i t 8.6: the Nu '-motive t~spiration Quite different inhibitor and activator patterns were r e v e a l e d w h e n the vesicles w e r e p r e p a r e d l r o m bacteria g r o w n at p H 8.6 (Figs. 3 - 5 ) . in Fig. 3 the d a t a for Bac. FTU a r c p r e s e n t e d . O n e can see that now C C C P is s t i m u l a t o r y . Such an effect was s h o w n in b o t h N A D I - t - - ~ f u m a r a t e a n d a s c o r b a t e --, O z s p a n s (Fig. 3A a n d B, C, D, respectively). T h e Na + u p t a k e a p p e a r e d to be rt_. :,tant to a m i l o r i d e (Fig. 3A, B) but s e n s i t i v e to A g " a n d E T H 157 {Fig. 3A. B). D E A a c e t a t e was ineffective in the a b s e n c e of C C C P and was s t i m u l a t o r y in its p r e s e n c e so that the r a t e o f the N a + u p t a k e in the s a m p l e s c o n t a i n i n g b o t h C C C P and D E A a c e t a t e r e a c h e d 0 - , ' :,; the s a m p l e s with v a l i n o m y c i n (Fig. 3C). It s h o u l d bc s t r e s s e d that d r a m a t i c d i f f e r e n c e bet w e e n the d a t a s h o w n in Figs. I and 3 was e n t i r e l y duc to t h e d i f f e r e n t p H v a l u e s of t h e g r o w t h m e d i a . In b o t h cases, the p H o f the i n c u b a t i o n m i x t u r e was identical and e q u a l t o 8.2. W h e n vesicles o b t a i n e d f r o m b a c t e r i a g r o w n at a l k a l i n e p H w e r e i n c u b a t e d at ncutr~,l pH, they still r e s p o n d e d in ' t h e a l k a l i n e f a s h i o n ' . Similarly. vesicles f r o m the " n e u t r a l b a c t e r i a ' , w h e n i n c u b a t e d at alkalit,e p H , b e h a v e d like t h o s e i n c u b a t e d at n e u t r a l pH. A n e x a m p l e o f this kind is s h o w n in Fig. 3D. H e r e the vesicles i s o l a t e d f r o m Bac. FTU g r o w n at p H 8.6 w e r e i n c u b a t e d at o H 7.5. O n e can see that C C C P is s t i m u l a t o r y r a t h e r t h a n inhibitory. M e a s u r e m e n t s o f t h e r e s p i r a t i o n rate r e v e a l e d that n e a r l y 4 0 % o f t h e total o x y g e n c o n s u m p t i o n by the vesicles f r o m the "alkaline bacteria" was sensitive to low [cyanide], t h e rest b e i n g i n h i b i t e d by m u c h h i g h e r

c y a n i d e c o n c e n t r a t i o n . R e s p i r a t i o n resistant to h , ~ [ c y a n i d e ] was absent f r o m t h e "n,:utral" b a e t c r i ; t (Fig. 4 A ) . t ~ a r a l l c l m e a s u r e m e n t s uf the N a " u p t a k e s h o ~ e d

that it v, as resiMant o r sensitive to low [cy'anide] in the "alkaline" or "neutral" b a c t e r i a , respectively (Fig. 4B). A g a i n . E. coli p r o v e d to r e s p o n d like Bac. FTU 5 ) . O n e e l t l l s e e that ( ' ( ' ( 7 I ) M i l n u l a t e s t h e N i l " u p t a k e s u p p o r t e d by e l e c t r o n t r a n s f e r f r o m N A I ) t t to 0 2 ( F i g . 5 A , B, ('), l r o m N A I ) t t t o l ' u m a r a t e (l:tg. 51))

(Fig.

a n d f r o m a s c o r b a t c to O , (Fig. 5E. F). In the first ease. I0 m M K e N c o m p l e t e l y i n h i b i t e d the N a uptake (5A). M o n e n s i n and A g " w,.'re also inhibitory (Fig. 5B) w h e r e a s a m i l o r i d e was w i t h o u t e f f e c t (Fig. 5('). I • 10" M H O N O which c o m p l e t e l y i n h i b i t e d the N a ' u p t a k e in the initial s p a n ~f the r e s p i r a t o r y chain (not s h o w n ) w a s i n e f f e c t ; v c in it,,, t e r m i n a l span (Fig. 51:).

Discussion T h u s , the study o n inside-out s u b e e l l u l a r vesicle~ , q Bac. F " 7 " as w e l l as E. coil i n d i c a t e s that the Ix, d c r h t g r o w i n g at n e u t r a l a n d a l k a l i n e p H e m p l o y e n t i r e l y d i f f e r e n t m¢'chanisms to e x p o r t N a " tenn. i.e., c ~ o p c r a tion of the H " - m o t i v e respiratotT chain and Na " / H ' a n t i p o r t c r in the f o r m e r case a n d the N a ' - m o t i v e r e s p i r a t o r y chair, in the latter. In t,oth | 1 - - and Na "m o t i v e r e s p i r a t o r y chains, two e n e r g y c o u p l i n g sites l o c a l i z e d in t h e initial and t e r m i n a l spans ~ t the c h a i n s w e r e i d e n t i f i e d (Fig. 6L it s h o u l d b e e m p h a s i z e d that the ~l|)ove ec, n¢lusions a r e c o n f i r m e d b~ all t h e n i n e lines o f e v i d e n c e listed in t h e m are actions o f such agenb, as

T a b l e !. A m o n g

15

16

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6

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Fig. 2, Na' uptake by the vesicles from E. cot/ grown at pit 7.5. Na" transport "~; • couplrd lo NAt)I! - O - electron ',r,,qqc~. Incuk=tkm mixture. 50 mM KzSO 4, 5 mM Na:S(_) 4. 3U mM MgSO 4. 5 mM potassium pho~ph,|t, liH) mM T~icinc-K{)tl. pll 7.75. fl.U, ~qh;~ncq. ak,,h~4 dehydrogcnasc (2 Uk 5- tO " M TPT and vesicles. 5 mg protein ml t Re.,piration ~',a, initiated b~ adding 5 m~,| N..XI)'. Add ti~,n,. I ili " M

valinomycin. 1 " ] ( I

~

M ('('('P. 2 mM amilofide mid 2fl mM DI:,X 4cetatL'.

100 C ( ' C ' P a n d I ) E A a c e t a t e , a f f e c t i n g t h e s y s t e m in t h e o p p o s i t e d i r e c t i o n s d e p e n d e n t o n w h e t h e r H +- o r Na'-motivc r e s p i r a t i o n s a r c involve," Effects of a m i l o r i d c , m o n c n s i n , A g + a n d low c y a n i d e a r c also demonstrative since these compounds specificity inhibit o n e o f t h e t w o N a " - t r a n s p o r t i n g m e c h a n i s m s s h o w n in Fig. 6. The Na~/H ~ antiporter-mcdiatcd m e c h a n i s m is q u i t e u s u a l f o r E. col;, ( f o r r e v i e w s , s e e R e f s . 16 a n d 17). A s to t h e primary, r e s p i r a t o r y N a + p u m p s , s p e c i ficly i n d u c e d u n d e r a l k a l i n e c o n d i t i o n s , t h e y w e r e o b s e r v e d in t h i s b a c t e r i u m for t h e first t i m e in o u r l a b o r a t o r y (fi~r p r e l i m i n a r y p u b l i c a t i o n , s e e R e f . 18). In t h i s r e s p e c t , E. coil p r o v e d to b e s i m i l a r to Bac. F T U a l r e a d y s l u d i e d in d e t a i l [9,10,12]. A d a p t a t i o n o f Bac. F T U a n d E. coli to t h e low [ H * ]

conditions seems to consist not only of the induction of the Na+-motive respiratory chain but also of the repression of the Na +/H + antiporter. The latter conclus i o n is b a s e d o n t h e f a c t s t h a t C C C P s t i m u l a t e s a n d m o n e n s i n i n h i b i t s t h e N a "- u p t a k e by v e s i c l e s f r o m t h e ' a l k a l i n e " b a c t e r i a . S u c h e f f e c t s w o u l d b e i m p o s s i b l e if t h e a n t i p o r t e r w e r e a c t i v e in t h e s e v e s i c l e s . O n t h e o t h e r h a n d , t h e H + - m o t i v e r e s p i r a t o r y c h a i n is, u n d e r t h e c o n d i t i o n s u s e d , still p r e s e n t in t h e Bac. F T U c e l l s grown at high pH. According to the data published e l s e w h e r e [12], it is t h e H + - c h a i n t h a t is r e s p o n s i b l e f o r s m a l l e r p o r t i o n o f o x y g e n c o n s u m p t i o n s e n s i t i v e t o low cyanide. The results of the present study can be compared w i t h t h o s e c a r r i e d o u t p r e v i o u s l y o n Vibrio aiginolyticus [ 3 - 6 . 1 q - 2 3 ] w h e r e N a ~- a n d H * - m o t i v e e n e r g y c o u -

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Fig. 3. Na * uptake by the "/eslcles from Bac. FTU grown at pH 8.6. Na ÷ transport was supported by NADH ~ fumarate (A) or ascorbate --* O z (B-D) electron transfers. For incuLation mixture in A, B and D, see Fig. 1, but n D pH was lowered to 7.5. In C, concentrations of Na2SO 4 and potassiuna ascorbate were increased up t,) 5 naM and 40 naM, respectively, whereas that of TMPD was decreased down to 0.25 raM, and 3.10 -5 M N,N'-dicyclohexyl carbodiinaide was used instead of TPT. Amiloride concentrations were 0.2 mM (A) and 1 mM (B). Vesicles, 5.2 (A), 3.3 (B), 3.7 (C) and 5.3 (D) nag protein nal- t. Other additions as in Fig. I.

101

O n the ,~:;*,,:r hand, the alkalophilic still isolated such as Bac. ahalopt'ih¢.s and Bac. firmus strains, studied by Krulwich and her colle"7,,os [24-27] usually grow at much lower [Na+]. Guffanti and Krulwich [24,25] failed to find any indications of Na * motive-respiration in the studied bacilli. Instead, the H + respiratory chain and N a + / H + antiporter were observed. W h e n the concentration of Na + outside the cell is rather luw, it is difficult to m a i n t a i n high /t~Na* required to support the Na+-driven A T P synthesis. So, the alkaline adaptation p r o b l e m should be solved in a way other t h a n induction of the Na*-motive respiratory chain. O n e of the possibilities seems to consist of organization of a 'third water space' isolated from both the cytoplasm a n d the outer medium. This might be done by formation of lenses in the cytoplasmic mere-

piing sites are present, i n d e p e n d e n t l y of the growth medium pH, at the initial and terminal steps of the respiratory chain, respectively. Thus, this bacterium is always well e q u i p p e d to live u n d e r either neutral or alkaline conditions. T h e very fact can easily be explained by the a d a p t a t i o n of V. alginolyticus to its natural niche, algal mat, where p H fluctuates (due to the photos: 'hetic activity of algae) from neutral in the m o r n i n g t~, ,'ine in the evening. It shoul, 9 t e d that for all the above microorganisms, Na ÷ ,_ ntration in the n a t u r a l e n v i r o n m e n t is rather high. ! t,.~ 0.5 M for V. alginolyticus (sea water) and a b o u t 0... M for E. coli (intestine). As for Bac. FTU, it is, according to microbiological a n d genetic tests, most probably a strain of the halotolerant Bacillus alcalophilus halodurans, surviving even in I M NaCI.

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Fig. 4. Cyanide inhibition of ascorbate oxidation (A) and the coupled Na + uptake (B) by the vesicles from Bac. FTU grown at pH 7.5 and 8.6. Incubation mixture, 0.1 M KzSO4, 5 mM NazSO4, l0 mM MgSO4, 50 mM Tricine-KOH, pH 8.2, 15 mM Tris-ascorbat¢, 0.5 mM TMPD, 4-10 -6 M TPT. The mixture was supplemented with 1-10 -5 M CCCP and 50 mM D E A acetate or with 1-10 -5 M valinomycin (growth at pH 8.6 or at pH 7.5, respectively). In A, the amount of vesicles was 0.5 mg protein m l - k In B, the am9unt of vesicles and the initial rate of Na + uptake without cyamde were 5.7 mg protein ml - t and 30 ngion (mg protein)- i rain - I (growth at ptt 8.6) or 6.2 mg protein ml-~ and 24 ngion (rag prot e i n ) - I rain T (growth at pH 7.5).

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Fig. 5. Na ' uptake h$ lhc scniclcn ol /-.. ~oh grown al p l l ,S.(). "lh¢ Na ' lralisporl ~as s u p p o r t e d h~, N A I ) I I ~ 0 2 ( A - ( ' k N A D I I --, f u m a t a t e (I)) a n d a % ' o r h a l c -- ():, (1{. F) def.-Iron lrlmsfcr,,, inct.l',alil)n n'fi~.lurc, 50 m M K :S()~, 5 m M N a : S ( ) l , 30 m M MgSO.~, 25 m M l~)lassium a c e l a l e (A). or p h o s p h a l c (('- [)), I00 m M T r i c m c - K ( ) l f , p l i 7.75. vc~,iclcs, 5 m g p r o l c i n ml L T h e m i × i u r c ~ a n s u p p l c m c n l c d with ().5c,:- e t h a n o l a n d a l c o h o l dt'l'Lvdrogcna~,c (2 U) ( l l - l ) ) . 5 ' 10 ~' M II:'T (('), IO m M K ( ' N (D). II) m M l " r i s - a s c o r h a t ¢ (E. F). T h e r e a c l i o n w a s s l a r l e d by a d d i n g 5 m M N A D t l (A). 5 m M N A I ) " ( l l - I)) o r it.5 m M r M F ' D ([:. F). O l h c r a d d i t i o n s . I • I0 5 M ( ' ( ' C P , l0 m M K ( ' N , I. I() s M Moncn.'dn, l • I(I ~ M A g N t ) ~ . 2 m M a m i l o H d e , 5 m M pola~,~.ium f u m a r a t c a n d I ~ I() ~ M l I O N ( ) .

I(13 Or'owTh o t p H 7.5

n a m e l y . ( i ) by grov, th at alkaline pt[. (it) i,y g r o ~ t h in t h e |'~lt_'~;Cl]CC ~,~l" a n tlllCOUl'~ler a n d

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Fig. (,, 1"~o mechanisms of Na ' export trom Bat, I"Tl' and t':" coil cells: c~peration of the I! '-motive respirat.rs, chain and N a '! I " ant|porter in cells gr~,wing ;it neutral pll tuppcr scheme) ;tlltl the Na "-motive respirator3 chain in cells gro~i0g a! alkaline pit IIo,,~cr ,,chcm¢ ),

p h o s p h o r y l a t i o n , a f u n c t i o n h a v i n g high t h r e s h o l d A-~, level [4,28]. It is i n ( c r e s t i n g that o t h e r m e m b r a n e - l i n k e d f u n c t i o n s h a v i n g n o such a high A ~ t h r e s h o l d , n a m e l y , s o l u t e i m p o r t s a n d motility, a r c N a ' - d c p e n , l c n t also in soil a l k a l o p h i l i c bacilli, b e i n g s e r v e d by N a ' . s o l u t e - s y m p o r t e r s a n d N a ' m o t o r , respectively. In t h e s e cases, A ~ . , . , u s e d as a d r i v i n g force, is p r o b a b l y p r o d u c e d by e l e c t r o g e n i c N a ' / H a n t i p o r t c r utilizing t h e H ~ r e s p i r a t o r y c h a i n - g e n e r a t e d .aq" to export N a ~ in spite o f the ~ppositely directed

apH [261. It is r e m a r k a b l e that such t a x o n o m i c a l l y distant s p e c i e s as E. coil a n d Bac. F T U e m p l o y i d e n t i c a l adaptation mechanisms consisting of induction of the a d d i t i o n a l r e s p i r a t o r y chain with two N a * - m o t i v e coupling sites and in repression o f the N a + / H * ant|porter. The very fact is indicative o f the wide distribution o f this type of adaptation a m o n g microorganisms. A l k a l i n e adaptation is hardly the only reason to have the Na ~ cycle. In our group, indications wcrc obtained that Na ÷ energeties could arise under neutral p H w h e n a p r o t o n o p h o r o u s uncoupler or 2 - 1 0 5 M cyanide were added to the growth m e d i u m . A s it was shown by Kakinuma et al. [29,31], N a * - A T P a s c of Streptococcus faecalis can be induced in three it, dependent ways. all of them resulting in A ~ H . decrease,

t h a t N a ' - A ' l ' P a s e o f S. l'aecali.~ d i l T c r s f r o m Paso a.~ ~ e l l as f r o m N a ' - A ' l P a s c o f . e . g . . P r o pion(~,cnum mo~h'.~tum w h i c h is o f l h e I:,,Ft l y p e [ 2 , 3 2 34],) noted II'-AI

At] i m p r e s s i o n arises thai b a c t e r i a m o n i t o r A ~ l ~ . level, r e s p o n d i n g to its d e c r e a s e by act(ration of t h e N a " cycle. Most p r o b a b l y , the r e a c t i o n to the A ~ , l o w e r i n g is not r e s t r i c t e d to such a r e s p o n s e . F o r e x a m p l e , in lhthd,a,'terium h a h , h i , m we :,;ho%%red that a d e c r e a s e in ..1~ I. results in a r e p e l l e n t signal c a u s i n g a c h a n g e in tile d i r e c t i o n o1' I l a g e l l u m r o t a t i o n . A ..lfiH. r e c e p t o r , c a l l e d " p r o t o m e t e r " ~,~as p o s t u l a t e d to be i n v o l v e d in such kinds o f e f f e c t s [35-3q]. A d a p t a t i o n to lhctors that lowor t h e ..l~ n level nla.v, a p p a r e n t l y , also i n d u c e s o m e systems o t h e r t h a n Na ~ c n c r g e t i c s , e s p e c i a l l y w h e n [ N a " ] is low and glycolytic s u b s t r a t e s a r e av~,ilablc ( l o t rcvica~s, see Refs. i7 a n d 40). H e r e too. i n v o l v e m e n t of t h e ..l~l t • r e c e p tor s e e m s p r o b a b l e . F u t u r e s t u d i e s will a n s w e r t h e q u e s t i o n as to what the role o f the N a " cycle is is] a d a p t a t i o n to the tow A ~ . conditions other than t h o s e u s e d in o u r study i,e. a e r o b i c gro~vth 1111 s u c c i n a t e at h i g h p H a n d high [Na ']. F o r E. c o h . g r o w t h at low . 1 ~ , ~ wa~ d e s c r i b e d ~vhcn g l u c o s e [411 or u n c o u p l e r s [42- 46] w e r e p r e s e n t .

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