Biochimica ut Bioph.vsica ,4eta, 4tH (1975) 440 457

C Elsevier Scientific Publishing (~ompan 3, Anlstcrdam

Printed in lhc Netherlands

BBA 77051

SUCCINATE TRANSPORT INORGANIC

IN B A C I L L U S

SUBTILIS.

DEPENDENCE

ON

ANIONS

(). K. GHEI AN[') WILLIAM W. KAY I)epartnlents ~/ Biochemi.~lrv and ,S'ur/le#'l'. L/#drervi:l O/ ,S'avl~atchewan. Sa.~Latmm. Sa,~l,atchewan S 7 N 0 1!'O ( ('ana¢ht I

I Rcccivcd .lantlar.~ 20th, 1975)

SUMMARY Cations were generally inefl'ective in stimulating succinate t r a n s p o r t in a succinate d e h y d r o g e n a s e m u t a n t o f B a c i l l u s s u h l i l i s unless a c c o m p a n i e d by polyvalent anions: p h o s p h a t e and sulfate being particularly active. The K,,, wilues l\~r the phosFhate or sulfate requirement were approx. 3 raM. giphasic kinetics were characteristic of both the succinate (h,,, values 0.1 and I m M ) , and inorganic p h o s p h a t e (K,, values 0.1 and 3 r a M ) t r a n s p o r t system(s). The phosphate t r a n s p o r t system(s) was repressed by. high inorganic p h o s p h a t e and a c o o r d i n a t e increase in the t r a n s p o r t o f phosphate, arsenate, and phosphate-sti mulatcd succinate t r a n s p o r t a c c o m p a n i e d growth in low p h o s p h a t e media. A class o1" arsenate resistant mutants were simultaneousl~ defective in the transport of arsenate, p h o s p h a t e and succinate when cells were repressed for phosphate transport, however, the t r a n s p o r t of these ions was regained in these mutant~, when grown in low phosphaite media. Organic p h o s p h a t e esters did not stimulate succinate t r a n s p o r t in arsenate resistant mutants but were effective after growth in low phosphate media. G r o w t h under p h o s p h a t e limitation permitted the simultaneous regain of both p h o s p h a t e and sulfate d e p e n d e n t succinate t r a n s p o r t activities wherea~ sulfate limitation alone was ineffective. Succ]nzite was not t r a n s p o r t e d by an anion exchange diffusion mechanism since p h o s p h a t e eJtlux was low or absent during succinate transport. The t r a n s p o r t of C¢-dicarboxylates in B. suhlili.v is strongly stimulated b\ i n t r a c d l u l a r polyvalent anions. The ab.,ence of an anion permeability mechanism precludes succinate t r a n s p o r t but partial escape from this restriction i~ mediated b \ the derepression of a p h o s p h a t e t r a n s p o r t system.

INTROI)U('TION M a n y bacteria utilize Krebs cycle carboxylic acids as sole sources o f c a r b o n and energy. However, the mechanism by which these metabolites are t r a n s p o r t e d and Abbreviation : H EPES. N-2-hydroxyethylpiperazine-N '-2-ethanesulphonic acid.

441 subsequently accumulated has not been extensively investigated. Theoretically, these substrates could enter the cell either as the uncharged protonated form at an unphysiologically low pH, or more likely as an ionized species at a higher pH. In order to transport anionic organic metabolites the cells must exercise compensating mechanisms to prevent unnecessary energy expenditure in forming both electrogenic and osmolar gradients. Such mechanisms presumably consist of either the cotransport of neutralizing equivalents of cations or the countertransport of excess anions from the cell. The well established mitochondrial di- and tricarboxylic acid transport systems appear to operate basically on at least three anion exchange systems [I, 2], two of which would be unlikely to function in bacteria due to inevitable carbon loss. Alternatively, there is the possibility that the counter-flow ion could in liict be an inorganic anion as in the mitochondrial L-malate-phosphate exchange system. In Bacillus subtilis the tricarboxylic acid cycle intermediates have been shown to be transported by three kinetically and genetically distinct systems which are specilic for the Ca-, C s- and C~,-tricarboxylic acids [3 5]. Competitive inhibition data has indicated that citrate [6] and the Ca-dicarboxylic acids [5] are most likely transported as anions. Recently it was demonstrated that citrate transport in an aconitase mutant of B. subtilis is markedly dependent on the presence of divalent cations and Ihat these cations are cotransported as a complex to preserve electroneutrality [7 ]. Succin,ate transport in B. subtilis has very unusual ion requirements relative to other metabolite transport systems in bacteria: that is that polywilent anions are predominantly required for effective transport, but the anion requirement is not specific. We decided to investigate further the nature of this requirement since it has important implications regarding the mechanism of succinate transport and the interaction of this transport system with other anion and cation transport systems. We suggest that succinate transport is largely dependent upon a general inorganic anion equilibrating mechanism in the absence of which the tricarboxylic acid cycle ex-dicarboxylic acids cannot be transported. EXPERIMENTAL

Or.qa/li.s'##ls Tile microorganisms used in this study were derivatives of the transformable strain B. subtilis 168, an indole auxotroph. B. subtilis I Aa22, is a succinate dehydrogenase-deficient ( s d h ) mutant [8], and strains 4-2B and 4-5B are mutants derived from I Aa22 which are resistant to 40 mM sodium arsenate. The succinate dehydrogenase mutants were routinely checked for purity by streaking on bromo-cresol purple indicator media [8]. The arsenate-resistant cells were also routinely checked for purity by streaking on low phosphate minimal agar and were routinely kept on nutrient agar containing 40 mM sodium arsenate. The isolation and characterization of strains 4-2B and 4-5B are described below. Stock cultures were kept either lyophilized or frozen in a medium containing 10 ?~i glycerol. 0.1 '~'opeptone and 20 mM L-malate. Media and culture methods Cultures were routinely grown on a New Brunswick gyratory shaker at 37 C in a minimal salts medium [9], to which sterile 0.1 ,~> peptone and 20 mM L-malate were added separately. The above medium was modified by the replacement of inorganic

442 phosphate by 0.1 M Tris. HCI (pH 7.0) and either 5 mM :~-glycerophosphate or 5 mM inorganic phosphate to obtain phosphate-limited cells. Sulfate-starved cells were obtained by growing cells in minimal medium in which MgSO, was replaced with MgCI 2, (NH,)2SO 4, with NH~NO 3 and 0.2 mM l-cysteine. Cells were routinely harvested from mid-exponential phase, washed three times with large volumes of either cold minimal rnedium or the appropriate phosphate- or sulfate-delicient medium, or 10raM Tris- HCI (pH 7.0) depending on the particular experiment. These cells were resuspended to I.I m g m l (dry wt), and kept on ice for a short time prior to the transport experiments. I.s'o]alion r~[arse#tale-resislaHl #Httlanl.s Cells of B. suhlilis I Aa22, the succinate dehydrogenase mutants, were grown on 0.5",, peptone, harvested and spread on nutrient agar plates containing 40 mM sodium arsenate. Spontaneous mutants which arose at an approximate frequency of 10 '-7 were isolated after incubation at 37 C l\~r 48, h, and were subsequently purified several times on the same media and checked for the parental genetic markers. This isolation procedure is essentially similar to that described for Escherichia coli b \ Medveczky and Rosenberg [10] and Bennett and Malamy [I I]. These strains were also routinely maintained on the same selection medium since they were found to undergo slow phenotypic reversion. ]~ra#ls/?orl assays Measurements of the transport of radioactive 2,3 -~ 4C-labeled succimite. "~sSlabeled sulfate, V'tAs-labeled arsenate, or 32p-labeled phosphate were perl\~rmed using the rapid filtration technique previously described [5]. Briefly, reactions were carried out in 5 or 10 ml reaction mixtures containing 10 5M radioactive substrates and I.I mg dry wt of cells per ml at 37 C. Cells were intermittently filtered through membrane filters (0.45 pin pore size: Sartorius), washed with 4 ml of the same incubation medium, dissolved in 5 ml of scintillation fluid (PCS, Nuclear Chicago) and assayed l\)r cellular radioactivity using a scintillation spectrometer (Nuclear Chicago. Mark II). Neither succinate, POt 3 nor AsO43 were chemically altered during transport as determined by autoradiography [10]. For the determination of transport kinetic data initial rates of transport were calculated from the linear uptake measurements at 15-s intervals l\~r 2 rain. Routinely boiled cell controls were done in all succinate transport experiments to avoid anomalous nonspecific binding or complex formation. In no instance was significant background binding found to occur. Chemicals 2,3-~ "~C-labeled succinate, 32P-labeled orthophosphate, "~5S-labeled sulfate and >~As-labeled arsenate were obtained from Amersham Searle Corp.

RESULTS Ion requirement ]br succinate trans7~ort Fig. IA illustrates the general ion requirements for succinate transport in B. subtilis I Aa22. Ceils resuspended in distilled water were unable to transport

443

--

2

o

E

E ¢-

¢./

tw

0

Ls

______o

B

O0 Z ,nr I--

o I.C

I,Ll I--

j'y~

Z 0

0..~

gO

0

2

4

6

8

10

MINUTES

Fig. I. Succinate transport into B. subtilis 1Aa22 in response to various cations and anions. Cells of the succinate dehydrogenase mutant previously induced for succinate by growth in the presence of 20 mM L-malate were washed (10 mM Tris - HCL pH 7) and incubated in various ion combinations at pH 7.0 for 10 min at 37 ' C prior to the addition of 2,3-~4C-labeled succinale (10 - s M, O.051tCi/ml). The cells were then quickly filtered and washed with the same buffer. The symbols in the graphs indicate the ion source present in the incubation and wash solutions. (A) Distilled water (11 I ) : 1 0 r a m T r i s - H C I ( O O ) ; 1 0 m M K2SO4 ( A A); 50raM KzHPO,~ ! KHzPO4 (O O ) and 5 0 m M K2SO4 ( A ~ ) . (B) 10raM Fez(SO4)s ( ~ U ) ; 1 0 r a m ZnSO4 (O O); 10raM FeSO4 ( A A); 10raM NazMoO4 ( O O ) and 1 0 m M MnSO4 ( ~ ,~-). Tris control (10 mM Tris- HCI) was the same as in Fig. [A.

succinate and very low transport velocities were observed in 10 mM Tris • HCI buffer (pH 7.0). Although low, this concentration of Tris - HCI buffer was used as a control suspension media because it effectively prevented autolysis and provided a low enough background o f succinate transport to compare the effects of added ions. Higher concentrations of Tris • HC1 (20 100 raM) were inhibitory to dicarboxylate transport. Potassium phosphate or potassium sulfate stimulated both succinate transport and accumulation in a manner which was concentration dependent. At 50 mM both phosphate and sulfate salts were highly effective (Table 1). Therefore, this system was not as ion-specific as has been frequently observed in various microorganisms for the transport of amino acids [12-17], carboxylic acids [18, [9] or sugars [20, 21 ].

444 I A BI.E I SU('('INATE

TRANSP()R'I

IN

TftE

PRESENCE

OF

VARIOUS

CATIONS

AND

ANIONS

B. suhti/i.s I A a 2 2 . a s u c c i n 4 t c d c h y d r o g e n a s c Illkll:411[ \\{l>, i n d u c e d for s u c c i n a l c t r a n s p o r t by gro~xth on miilinlal n l e d i u n l atld 20 filM I - m a l a t c . Cells w e r e \ \ a s h e d in 10 m M I r i s HCI b u f f e r ( p H vail a n d r c s t l s p c n d c d (1. I m g d r y \\1 m l ) in t h e s a m e bul'l'er a n d p r e i n c u b a t e d \ \ i t h x a r i o u s s:llt s o l u t i o n s tit 50 m M final c o n c e n t r a l i o n ul p H 7.0 f o r 10 rain. 2.3- ~"~('-Iabelcd s u c c i n a l e ( 10 " M. 0.05 ,,~('i ml) t r a n s p o r t ~\as Ihen i n c a s u r c d {tl v a r i o u s l i m e l n t c r ~ a l s for l0 rain. D a t a {ire e x p r e s s e d as r c l a t i x c initial

rate o1" s u c c i n a l c

Salt : l d d c d

linal aCctlnlulation

R c l a t i', e i n i t i a l

Rch.tti\ e

I'UIC

{ICCLInltl]tlt iOI1

1.0 1.3 1.0 0.1 4.0 KtI2P()¢

N a 2 H PO.~ , N a H 2 P O ¢ N a 2 H As()., K _,SO,, Ntl2g() 4 ( N [ [4 }2N()4 1-i2S()4 MgS()¢ ('tiN()4 K eC'r z()v I%tassium acetate Polassitlm

{lilt[ tiN, r c l a l i v e

{ll IO lllill.

.%lice ill4[e [ G.II1%pOl-[

None K('I NaCI NHaCI KN()s K2HP()a

transporl

{iscor b a l e

Potassiunl gluconate

11).7

6.S 6.