Transport of C4-dicarboxylic acids in Salmonella typhimurium

ARCHIVES

Vol.

OF BIOCHEMISTRY

AND

190, No. 1, September,

Transport

BIOPHYSICS

pp. 281-289,

1978

of C4-Dicarboxylic WILLIAM

Department of Biochemistry

W. KAY’

and Microbiology, Received

Acids in Salmonella

July

typhimurium

M. J. CAMERON

AND

University of Victoria, Victoria, British Columbia, Canada

26, 1977; revised

October

3, 1977

Cd-Dicarboxylic acids are transported into Salmonella typhimurium by stereospecific systems of both high and low affinity. Succinate and L-malate are accumulated in a tricarboxylic acid cycle mutant as was D(+)-malate in induced wild-type cells. Accumulated dicarboxylates are exchangeable with exogenous dicarboxylates. The trichloroacetic acid cycle dicarboxylates are the best inducers of their own transport. Specific mutants devoid of dicarboxylate transport activity (dct) were isolated and differed from tricarboxylate transport mutants (tct) with respect to growth and transport. A mutant devoid of oketoglutarate dehydrogenase was unable to transport dicarboxylic acids but citrate transport remained unaffected. Tricarboxylic acid cycle mutants were markedly dependent on an exogenous energy source for the transport of succinate, proline, or leucine. Dicarboxylate transport was largely inhibited by various metabolic inhibitors but could only be inhibited by N,N’-dicyclohexylcarbodiimide anaerobically. ATPase mutants were unimpaired in their ability to transport succinate or proline aerobically.

Microbial growth on various di- and tricarboxylic acids has been considered for some time to be dependent upon the synthesis of specific permeation systems (l-5). Recent interest in the transport of tricarboxylic acid (TCA)2 cycle intermediates has resulted in a higher resolution of the molecular events involved in the transport of the C4-dicarboxylic acids. In general a single system, albeit perhaps subdivisible into many separate components, appears to be a general occurrence in microorganisms (6). These systems have common properties of substrate specificity, active transport, and regulation by induction and repression (7-15); however the Bacillus subtilis (10, 11) and Escherichia coli (15) systems have been resolved to finer detail by the use of suitably blocked mutants or with a nonmetabolizable analogue (16). In E. coli, at least three genes have been implicated in

the transport process (8, 15, 17); however, the precise gene products have yet to be determined. The active transport of succinate has been studied in membrane vesicles of E. coli (6, 18, 19), and recent work (20) suggests that this system may be atypical of other metabolite transport systems in membrane vesicles. In this study we characterize the dicarboxylate transport system in SaZmoneZZa typhimurium succinic dehydrogenase mutants and also with the use of the nomnetabolizable analogue n (+) -malate. Some studies in nonblocked strains were recently reported (21). MATERIALS

AND

METHODS

Organisms. All microorganisms used in this study were derivatives of S. typhimurium LT2 and are listed in Table I. The strains were maintained in nutrient agar stab cultures or lyophilized and stored at -20°C. Cells were routinely grown on minimal salts media (22) or on nutrient both with the carbon source added separately. Cultures were incubated in 50-ml vol at 37°C in 250-ml flasks in a gyratory shaker to a cell density of approximately 5 x 10” to lO’/ml. TCA cycle mutants. All TCA cycle mutants used in this study were isolated as colonies unable to grow on citrate (10 mM), succinate (20 mM), L-malate (20

’ To whom all correspondence should be sent. ’ Abbreviations used: TCA, tricarboxylic acid; fluorocarbonylcyanide phenylhydrazone; FCCP, CCCP, carbonylcyanide 3chlorophenylhydrazone; DNP, 2,4-dinitrophenol; p-CMB, p-chloromercuribenzoate; NEM, iV-ethylmaleimide; DCCD, N,N’-dicyclohexylcarbodiimide; HOQNO, 2-n-heptyl-4-hydroxyquinoline-N-oxide. 281

0003-9861/78/X)1-0281$02.00/O Copyright D 1978 by Academic All rights

of reproduction

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in any form

reserved.

282

KAY TABLE

STRAINS

OF S. typhimurium

Strain designation

:%s’

I USED IN

THIS

STUDY"

Origin

LT2 HfrA c5 AC34 Dl LT2.FM2 HfrA.FC

AND

4

Wild-type Wild-type SDH, MDH ACN KDH DCT TCT

Laboratory stock K. Sanderson (Calgary) Derived from HfrA

CAMERON ration. 3-Fluoromalate was the generous gift of P. Kent, University of Oxford. n(+)-[:‘H]Malate was synthesized from n-[“Hlaspartate via 2-[:‘H]bromosuccinate (29) by mild alkaline hydrolysis. Both bromosuccinate and n(+)-malate were purified by preparative thin-layer chromatography (28). All other chemicals, analogues, and inhibitors were purchased from Sigma Chemical Company. RESULTS

Derived Derived Derived Derived

a Abbreviations used are: SDH, genase; MDH, malic dehydrogenase; KDH, a-ketoglutarate dehydrogenase; ylate transport; TCT, tricarboxylate

from from from from

HfrA HfrA LT2 HfrA

succinic dehydroACN, aconitase; DCT, dicarboxtransport.

mM) minimal medium after nitrosoguanidine induction (23), and penicillin enrichment in the same minimal medium (24). Mutants were screened for histidine auxotrophy and acid secretion (25) also. This same procedure was also used to isolate ATPase defective strains. Transport negative mutants. Dicarboxylate transport mutants (dct A) were isolated as spontaneous mutants resistant to 3-fluoromalic acid (17) on pyruvate (20 mM) minimal medium. Spontaneous mutants were purified and checked for the negative growth phenotype on dicarboxylate minimal media. Citrate transport mutants were isolated as spontaneous fluorocitrate resistant clones on fumarate minimal medium and checked for citrate transport and the original histidine auxotrophy (26). ATPase mutants were isolated after nitrosoguanidine induction as strains pleiotropically defective in the utilization of acetate; citrate and L-malate are sole carbon sources. All mutants isolated from S. typhimurium were assayed for all TCA cycle enzymes by standard assay procedures as previously reported for E. coli (27). Transport assuys. Transport assays with labeled dicarboxylic acids or amino acids were carried out by rapid membrane filtration as previously described (27). Total radioactivity of the assay mixtures was determined from remaining radioactivity after acidification of the reaction mixture with 1 N HCI. Transport assays with succinate were routinely performed using the succinic dehydrogenase mutant C5. Chromatography. To determine the fate of radioactive succinate, cells (1 ml) were incubated for the desired time with 10 mu succinate (0.05 pCi/pmol), ffltered, washed as usual, and extracted with ice-water/toluene as previously described (7). The contents were lyophilized, resuspended to 0.1 ml and chromatogramed by thin-layer chromatography (28) and analyzed by radioautography. Chemicals. All labeled dicarboxylic acids and proline were purchased from Amersham/Searle Corpo-

Induced cells of S. typhimurium not harboring a genetic lesion, in the enzymes of the TCA cycle appear to take up labeled succinate from the medium rapidly (Fig. 1). Autoradiography of the intracellular contents, however, revealed that succinate was rapidly metabolized and not significantly accumulated in wild-type strains. However, strain C5, a double mutant deficient in succinic dehydrogenase and malic dehydrogenase took up succinate more slowly, but accumulated both succinate and L-malate intracellularly to levels of approximately 20and 60-fold, respectively (Fig. 2). Figure 2 also depicts the time course of the accumulation of intracellular dicarboxylic acids. Mutants defective in the ability to transport citrate (FC4) were similar to the wildtype induced cells with respect to succinute transport, thus genetically and functionally differentiating the dicarboxylic and tricarboxylic transport systems in this microor-

0

2

4

6

8

10

TIME (min) FIG. 1. Succinate uptake into induced wild-type and mutant strains of S. typhimurium. The strains were grown in succinate (15 mu) nutrient broth (SNB). [2,3-%]Succinate (10~~) uptake was followed in washed cells in the presence of 5 mM cu-glycerolphosphate. o--O, Wild-type; Cl---U, FCR4; A-A, C5; A-A, Dl; and &--O, FM2.

C,-DICARBOXYLATE

I

283

TRANSPORT

0-o-c

0

Malate

/

Succinate .-•

.A

.’

0

2

4

TIME

6

8

l(

(min)

FIG. 2. Time course of dicarboxylate pool formation in S. typhimurium C5. Washed, induced cells we& incubated with 5 mMa-glycerolphosphate and [2,3-“%]succinate (10 pM) for various intervals, then filtered, washed, and extracted in toluene-water. The radioactive dicarboxylates were separated by thinlayer chromatography, analyzed by autoradiography, and the appropriate labeled material was removed and assayed by scintillation spectrometry. The level of accumulation was calculated from the known specific activity of succinate and the intracellular water volume (4 &mg).

ganism (Fig. 1). Also mutants (FM) isolated as spontaneous 3-fluoromalate resistant organisms were largely defective in the uptake of labeled succinate. (Fig. l), in agreement with previous reports on E. coli (15, 17) and S. typhimurium (17, 21). These latter mutants are of the dct A variety as reported previously (21); this was confirmed by genetic mapping; these mutants have been closely linked to the xyl gene (unpublished results). Of particular interest was the observation that various mutants defective in a-ketoglutarate dehydrogenase (EC 1.2.4.2) were nearly devoid of dicarboxylic acid transport activity. These were not the results of multiple mutations since revertants simultaneously regained both enzyme and succinate uptake activity. A similar situation has been reported in B. subtilis (11).

Kinetics

of Dicarboxylic Acid Transport When the transport of the Gdicarboxylates, succinate, fumarate, L(-)-malate, and D(+)-malate were studied as a function of substrate concentration, biphasic kinetics were observed with all substrates with the exception of D(+)-malate. Figure 3 demon-

FIG. 3. Kinetics of succinate and fumarate transport into S. typhimurium C5 as a function of dicarboxylate concentration. Induced, washed cells were added to isotopic reaction mixtures to initiate the transport experiment. Rate data were calculated from linear transport rates obtained by 15-s sampling up to 1 min. Data are plotted by the Eadie-Hoffstee method.

strates clearly the biphasic system for succinate and fumarate on an Eadie-Hoffstee plot. The kinetic constants are listed in Table II. With the exception Of D(+)-malate all of the dicarboxylic acids exhibit apparently both high and low affinity transport kinetics differing in apparent affinities for the substrates by an order of magnitude. The affinities described by these kinetics are succinate > L-malate ‘r. D(+)-malate; however, the lower affinity kinetics are Lmalate > succinate > fumarate. No low affinity system was detected for D(+)-malate as the transport at high substrate concentrations was impossible to separate from diffusion.

Competitive Inhibition Acid Transport

of Dicarboxylic

The inhibition of succinate transport by various analogues in mutant C5 was generally found to be specific for Gdicarboxylates (Table III), among which the TCA cycle dicarboxylates were the most competitive-with the exception of oxalacetate. Neither cY-ketoglutarate nor citrate was greatly competitive. This agrees with the result of the citrate transport mutant (Fig.

284

KAY TABLE

AND

II

KINETIC CONSTANTS FOR DICARBOXYLATE TRANSPORT IN INDUCED CELLS OF S. typhimurium” Substrate Succinate Fumarate L-MaIate D-MaIate

V nlax

K?lt (M) 1) 2) 1) 2) 1) 2) 1)

1.4 1.8 2.1 5.0 1.9 1.0 2.0

x x x x x x x

bun min-’ 10-5 W4 10-5 w 10-5 1o-4 10-5

mg-‘)

6.7 17.4 9.8 33.2 6.6 20.1 3.6

a Washed induced cells of strain C5 were assayed for dicarboxylate transport at variable substrate concentrations. Reactions were initiated by the addition of cells to a final concentration of 0.35 mg dry weight ml-’ and samples withdrawn at 15-s intervals for 1 min to determine initial rates. Kinetic parameters were calculated directly from double reciprocal plots of the rate data.

1) and with some previously published results (21). A wide variety of analogues also were tested for the inhibition of succinate uptake (Table IV). From a comparative analysis of these, it can be concluded that any large group modifications preclude recognition by this system. Also carbon chain shortening, even to a minor degree as in acetylene dicarboxylate, is not favored nor was chain lengthening (glutamate). The system is not strongly stereospecific since n (+) -malate is both transported and a competitive inhibitor. Even small substitutions at position three in the carbon chain greatly reduce the efficacy as competitive inhibitors. The most effective competitive inhibitors were 2-bromosuccinate, monomethylsuccinate, succinamate, and acetylene-dicarboxylate. Interestingly, a single mod& cation of a carboxyl doesn’t prevent competitive inhibition but modification of both groups precludes recognition. A similar observation has been made for B. subtilis ( 11). From the kinetics of competitive inhibition it was observed that the relative affinities for the various substrates were in general higher for the physiological C~-dicarboxylates than for the analogues. Difficulty in getting good competitive inhibition data at higher concentrations was encountered and is not presented. The Ki values for inhibition are in general agreement with the K, values for uptake (Tables II and IV).

CAMERON

Exchange of Dicarboxylic Acid Pool When intracellular dicarboxylic acids were allowed to accumulate either in mutant C5 or with n(+)-malate the labeled material was freely exchanged for unlabeled exogenous dicarboxylates (Fig. 4). Rapid exchange occurred, particularly up to 5-10 min. Thus, the transport system facilitates rapid exchange between the exogenous and endogenous material. Like the uptake results the exchange was particularly selective for the TCA cycle dicarboxylates (Table V). Induction of Dicarboxylate Transport When strain C5 was grown on pyruvate in the presence of various dicarboxylates a variation in the ability to transport succinate was observed. A comparative experiment with the wild-type strain grown in nutrient media in the presence of dicarboxylates and assayed for n(+)-malate transport produced similar results; that is that TCA cycle C4-dicarboxylates were the best inducers of the transport process (Table VI). It is difficult a priori to decide which of these is the true inducer due to metabolism. This conclusion was strengthened by the observation that other dicarboxylate analogues, notably bromosuccinate also gratuitously induced transport. As in E. coli (7, 15) the transport of dicarboxylic acid was markedly repressed by glucose (21). Energization of Dicarboxylate Transport in TCA Cycle Mutants It is now well documented that a marked requirement for an energy source is characteristic of metabolic transport in membrane vesicles. However, this is an infrequent occurrence in whole cells unless drastically poisoned (30). We have found in S. typhimurium that the efficiency of transport of succinate, proline, or leucine is greatly stimulated by an exogenous energy source, especially in various TCA cycle mutants. In Fig. 5 the relative effect of an aconitase mutation on transport is demonstrated. Whereas some stimulation occurs in the C5 mutant (and none in the parent strain) a marked dependency on energy source was observed in this aconitase mutant. A similar dependence on energy

Cd-DICARBOXYLATE TABLE COMPETITIVE

INHIBITION

AnalOgW

OF DICARBOXYLATE

Inhibition succinate

Succinate Fumarate L-Malate L-Aspartate Oxaloacetate a-Ketoglutarate Citrate n-Aspartate Bromosuccinate Citraconate Citramalate Glutarate tn.-erythro-/3-hydroxyaspartate DL-threo-/3-hydroxyaspartate Itaconate @Malate Maleate Malonate Mesaconate Mercaptosuccinate cY-Methylsuccinate Monomethylsuccinate Succinamate

of uptake

III TRANSPORT

(8)

TABLE

Inhibitor Fumarate L-MaIate L-Aspartate Bromosuccinate Monomethylsuccinate Succinarnate Acetylenedicarboxylate

BY ANALOGUES

IN S. tvohimurium”

Analogire

DC+)Malate

ML.?

97 97 90 71 10 53 16 6 79 0 4 7 2

91 92 95 24 15 71 26 6 84 1 0 5 3

67 87 90 87 0 4 22 17 26 13

59

-

-

12 42 15 10 30 25 19 66 63

46 3 18 39 20 9 75 54

80 13 26 3 2

a CeIIs of strain C5 previously induced for dicarboxylate (10 FM) in the presence of 1 mM unlabeled analogue. dicarboxylate was calculated from initial rates determined

COMPETITIVE INHIBITION IN S. typhimurium

285

TRANSPORT

IV

OF SUCCINATE TRANSPORT BY DICARBOXYLATES~

“*

Inhibition

(%)

cinate

LMalate

DC+)Malate

50 26 9 6 6 54 12 53 21 0 0 1 0 65 3 0 39 5 10

27 10 18 0 12 46 25 33 24 2 10 7 0 56 0 0 31 6 0

17 17 0

9 6 8 2

10 4 11 8

-

SUC-

D(-)-Tartarate L-(+)-Tartarate Meso-Tartarate Succinamide N-Hydroxysuccinimide Phenylsuccinate p-Nitrophenylsuccinate 3-Nitropropionate Bromomaleate 1,4-Butanediol 3-Chloropropionate Glutaconate Adipate Acetylenedicarboxylate Succinonitrile Fumaronitrile 2,3-Dibromosuccinate Iminodiacetate 1,3-Acetonedicarboxylate Dimercaptosuccinate Butyric acid Glutamic acid Fumarate dimethyl ester

of uptake

transport were assayed for dicarboxylate transport Percentage of inhibition of uptake of radioactive from 30-s samples for 2 min.

dehydrogenase (unpublished results) in agreement with that previously demonstrated for E. coli (13).

Kz (Mb 1.9 2.3 4.8 1.6 4.2 6.3 3.8

x x x x x x x

lo1O-5 W5 1O-4 1O-4 IO-’ 1O-4

0 Induced cells of strain C5 were assayed for [2,3-‘%]succinate uptake at 10 pM in the presence of four concentrations of unlabeled inhibitor. Initial rates were determined from filtration data obtained at 15-s intervals for 1 min. K, values were calculated from the resulting plots of the slope of the saturation curve versus inhibitor concentration.

source can be demonstrated in a mutant with a double TCA cycle mutation at succinic dehydrogenase and a-ketoglutarate

Inhibition of Succinate abolic Inhibitors

Transport

by Met-

A comparison of succinate uptake into the aconitase mutant AC34 energized with n(-)-lactate and C5 energized with cu-glycerolphosphate is shown in Table VII. In a general sense, the effects of metabolic inhibitors were the same in both strains. Agents usually described as proton conductors (FCCP, CCCP, and DNP) greatly inhibit transport. Inhibition of electron transport also (CN, azide, Amytal) inhibited transport, indicating that respiration is required for succinate transport. Ion gradients or ion permeability may also be of importance since the ionophores gramicidin and valinomycin were also inhibitory. The

286

KAY AND CAMERON TABLE

VI

UPTAKE IN S. typhimurium” Added compound Relative rate of transport of

INDUCTION

OF DICARBOXYLATE

succinate*

TIME

(min)

FIG. 4. Exchange of the dicarboxylate pool in S. typhimurium C5. Induced cells were incubated with [2,3-‘4C]succinate for 10 min (A) or with D(+)cH]malate for 2 min (B) prior to the addition of 1 mM unlabeled C$dicarboxylate. Cells were then filtered at various intervals and assayed for remaining radioactivity. A-A, Oxaloacetate; M, fumarate; o---O, L(+)-malate. TABLE EXCHANGE

V

OF ‘Y!-DICARBOXYLATE

POOL IN

tvdimurium a Analogue added (1 mru) Pool (8) Exchanged min from

S,

<1

suc&ate Succinate Fumarate L-Malate n-Malate Oxaloacetate Citrate L-Aspart&

n-Aspartate Bromosuccinate Succinamate

61 85 79 9 0 66 -

in 10

D(+)Malate 77 87 88 91

-40b -12 84 0 -5 42

DInduced cells were incubated with 10 p [2,3-“Clsuccinate for 10 min to establish the intracellular dicarboxylate pool. The D(+)-malate pool was established at 2 min. Equal aliquots of labeled cells were dispensed into preincubated vessels containing the unlabeled analogue to give a 1 mM final concentration and cells were fiitered at various intervals. b Cells continued to take up label.

effect of sulfhydryl group inhibitors is perplexing. Although p-CMB, iodoacetamide, and NEM were inhibitory to succinate transport in a-glycerolphosphate energized cells (US), no effect was observed in D(-)lactate energized cells (AC34), suggesting that the former inhibition may occur at a site close to the primary dehydrogenase. Succinate transport was sensitive to an-

None

1.0

Succinate

3.9

Fumarate L(+)-Malate

4.7 3.0

D(+)Malate’ 1.0 2.1 2.8 3.7

D(-)-Mdate

1.0

1.0

L-Aspartate n-Aspartate Maleate Mercaptosuccinate o-Methylsuccinate Succinonitrile Oxaloacetate Bromosuccinate Dibromosuccinate Mesaconate Monomethylsuccinate Succinamate n(-)-Tartarate Dimercaptosuccinate Acetylenedicarboxylate Glucose

2.1 1.8 1.9 1.3 2.5 1.2 1.1 2.7 1.9 1.0 2.0 2.3 2.2 1.6 1.8 0.15

2.3 1.4 2.3 1.0 1.2 1.0 1.0 1.2 1.3 2.3 1.0 1.4 0.16

‘Cells were grown on pyruvate (20 mM) minimal medium, which gives a partially induced state, and the inducers were added to this at 5 mu final concentrations. Washed cells were then assayed for the transport of succinate or D(+)-mahte at 10 PM. * Succinate transport was measured in strain C5. ’ n(+)-Malate transport was measured in HfrA.

aerobiosis but not completely (Fig. 6). Similar inhibition patterns were observed in response to the ATPase inhibitor DCCD. However, anaerobically in the presence of DCCD, succinate transport was greatly reduced, suggesting that both ATP and respiration can serve as routes of energizing succinate transport (30), presumably via the proton gradient.

Succinate

Transport

in ATPase

Mutants

Several ATPase mutants were isolated in order to ascertain whether the ATPase was as absolute a requirement for succinate transport in S. typhimurium as reported for E. coli (20). From the results in Table VIII it can be seen that of three such mutants examined essentially no adverse effect on aerobic succinate or proline transport was

C,DICARBOXYLATE

287

TRANSPORT TABLE

VII

INHIBITION

OF SUCCINATE TRANSPORT IN S. typhimurium BY METABOLIC INHIBITORS”

Inhibitor

Concentration (Ml

Inhibition succinate AC34 b

TIME

(min)

FIG. 5. Energixation of succinate, proline, and leutine transport in S. typhimurium strains AC34 and C5. Washed cell suspensions of strains AC34 (A-C) and C5 (D-F) were incubated in the absence (M) or presence (0--O) of 5 mru a-glycerolphosphate for 10 min prior to the addition of 10 PM [2,3-‘%]succinate (A and D), 1 pM [3H]proline (B and E), or 1 PM C3H]leucine (C and F).

incurred, even with cells drastically reduced in ATPase activity (E53). All these mutants had the phenotypes reported for the E. coli ATPase mutants; that is they were unable to grow on TCA cycle intermediates, but were able to oxidize them, and had reduced molar growth yields anaerobically. DISCUSSION

C4-Dicarboxylic acids are transported in S. typhimurium, apparently, by a transport route of substrate specificity wide enough to encompass the TCA cycle Qdicarboxylates. The specificities are nearly identical with those observed for E. coli (6,7,12,15), Pseudomonas (15), B. subtilis (10, ll), and even a marine diatom Cylindrotheca fusiformis (31). Thus, this transport system appears to be rather ubiquitous among microorganisms. The substrate specificities appear to be nearly identical with E. coli; however, what is novel is the demonstration of biphasic transport kinetics. These kinetic plots, which in many cases are attributed to the interaction of separate systems with

FCCP FCCP CCCP CCCP HOQNO KzFeCN, Azide Axide Amytal Amytal Oxamate Valinomycin Valinomycin Iodoacetamide Iodoacetamide Arsenite Arsenite PCMB NEM DNP DNP KCN KCN

W4 1o-3 1o-5 W4 10-4 lo-* 10-3 1o-2 1o-3 1o-2 1o-2 0.1 mg/ml 1.0 mg/ml 10-3 lo-* 1o-2 10-l 1o-4 1o-4 1o-4 1o-3 10-3 10-z

86 96 86 96 15 9 32 a2 -32d -17 -6 0 89 -5 -3.5 53 96 -5 -3 9 85 41 64

(W) of uptake C5’ 39 65 85 88 63 43 47 85 94 35 1 71 23 93 50 3 77 -

n Induced cells of strain AC34 or C5 were incubated for 15 min in the presence of the inhibitor prior to the assay Of SuCCkite uptake at 10 pM. ’ Energized with n(-)-lactate (5 mM). ’ Energized with a-glycerolphosphate (5 mM). d Cell continued to take up the label.

overlapping substrate specificities as in the case of aspartate transport in E. coli (27), can reasonably be interpreted as a low affinity form of the transport protein(s). Indeed, efflux measurements from E. coli vesicles (20) have indicated a low affinity exit reaction. Whether this is due to a low affrnity form or to another system remains to be seen. Transport kinetic data from E. coli also show biphasic plots in both whole cells and membrane vesicles (Kay, Kornberg, and Dawson, unpublished results); however, they are not as distinct as that demonstrated here for S. typhimurium. The obvious advantage of multiple systems or a system with these kinetics is the efficiency of transport over a wide substrate concentration range. The isolation of distinct dicarboxylate

288

KAY AND CAMERON TABLE TRANSPORT

Strain

OF SUCCINATE

VIII BY ATPASE

S. typhimurium” ATPase (o/o)

MUTANTS

Uptake’ succinate 9.1 10.7 11.1 8.6

TIME

(min)

FIG. 6. Effect of DCCD and anaerobiosis on succinate transport by S. typhimurium C5. Induced cells were incubated with 5 mu glucose for 10 min prior to the addition of 10 w [2,3-‘%]succinate. Anaerobic transport was measured with ceIIs in sealed nitrogen flushed vessels. M, Control aerobic cells, A-A, anaerobic cells; G-0, aerobic cehs and 1 mM DCCD; A-A, anaerobic cehs and 1 mM DCCD.

and tricarboxylate transport mutants genetically separates these systems and reinforces competitive inhibition data. An interesting finding that mutant Dl devoid of a-ketoglutarate dehydrogenase was also unable to effectively transport succinate suggests that this strain is perhaps unable to form dicarboxylate inducers; however, it is equally possible that repression is mediated through the accumulation of a-ketoglutarate. We are unable to differentiate between these alternatives as yet, although an identical effect was observed with B. subtilis mutants and has been explained on the basis of inhibition of the transport system (32). Competitive inhibition by various analogues can provide information on the substrate specificity of the transport system; however, inhibition does not of necessity mean the analogue itself is accumulated or even very permeable. Studies with 2-bromosuccinate suggest that it is not trans-1 ported since it is unable to displace the dicarboxylate pool. Also, experiments with labeled 2-bromosuccinate suggest that this analogue is impermeant (unpublished resuits) . One of the most intriguing aspects of metabolic transport is the mobilization of energy to effect active transport. Although

OF

Proline 0.41 0.37 0.53 0.23

HfrA 100 6.5 E 13 E 52 4.5 0.2 E 53 cIInduced cells of the various strains were assayed for succinate uptake as usual and the degree of uptake was calculated at 10 mm. b Succinate transport (nmoI/mg/lO mm) at 10 pM; prohne transport (mDOl/mg/lo min) at 1 pM.

membrane vesicle work has indeed been monumental in the understanding of energization via respiration (33-36), it is also apparent that ATP can also be used to energize transport (30, 37). In whole cells both these routes of energization appear to operate with respect to succinate transport. Thus succinate is still transported anaerobically in the absence of electron transport, is inhibited by DCCD, and finally is largely inhibited by a combination of these conditions. Three ATPase mutants, however, appear to be normal with respect to aerobic succinate transport, suggesting that respiration does play the major role in this regard. A priori it would seem reasonable that energy generation via the TCA cycle and respiration would be of ultimate importance to cells growing on TCA cycle intermediates such as succinate. We are unable to explain the difference recently observed with E. coli (20) that ATPase mutants were unable to take up succinate, particularly in view of the demonstration that succinate active transport in E. coli appears to be in response to the transmembrane proton gradient (38). REFERENCES 1. CAMPBELL, J. J. R., AND STOKES, F. N. (1951) J. Biol. Chem. 190,853-858. 2. BARRETT, J. T., AND KALLIO, R. F. (1953) J. Bacterial. 66, 187-192. 3. KOGUT, M., AND PODOSKI, E. P. (1953) Biochem. J. 55, 800-811. 4. HARVEY, R. J., AND COLLINS, E. B. (1962) J. Bacterial. 83, 1005-1009. 5. CLARKE,

P. H., AND

Gen. Microbial.

MEADOW,

20, 144-155.

P. M.

(1959)

J.

C,-DICARBOXYLATE 6. MATIN,

A., AND KONINGS,

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