JOURNAL OF BACTERIOLOGY, July 1991, p. 4411-4416

Vol. 173, No. 14

0021-9193/91/144411-06$02.00/0 Copyright © 1991, American Society for Microbiology

Bioenergetic Consequences of Catabolic Shifts by Lactobacillus plantarum in Response to Shifts in Environmental Oxygen and pH in Chemostat Culturest CHING-PING TSENG,1 JYA-LI TSAU,2 AND THOMAS J. MONTVILLE'2* Department of Food Science2 and Graduate Program in Microbiology,' New Jersey Agricultural Experiment Station, Cook College, Rutgers-The State University, New Brunswick, New Jersey 08903 Received 15 January 1991/Accepted 13 May 1991

Proton motive force (PMF), intracellular end product concentrations, and ATP levels were determined when a steady-state Lactobacillus plantarum 8014 anaerobic chemostat culture was shifted to an aerobic condition or was shifted from pH 5.5 to 7.5. The PMF and intracellular ATP levels increased immediately after the culture was shifted from anaerobic to aerobic conditions. The concentrations of intracellular lactate and acetate, which exported protons that contributed to the proton gradient, changed in the same fashion. The H+/lactate stoichiometry, n, varied from 0.8 to 1.2, and the H+/acetate n value changed from 0.8 to 1.6 at 2 h after the shift to aerobic conditions. The n value for acetate excretion remained elevated at aerobic steady state. When the anaerobic culture was shifted from pH 5.5 to 7.5, intracellular ATP increased 20% immediately even though the PMF decreased 50% as a result of the depletion of the transmembrane proton gradient. The H+/ lactate n value changed from 0.7 to 1.8, and n for H+/acetate increased from 0.9 to 1.9 at pH 7.5 steady state. In addition, the H+/acetate stoichiometry was always higher than the n value for H+/lactate; both were higher in alkaline than aerobic conditions, demonstrating that L. plantarum 8014 coexcreted more protons with end products to maintain intracellular pH homeostasis and generate proton gradients under aerobic and alkaline conditions. During the transient to pH 7.5, the n value for H+/acetate approached 3, which would spare one ATP.

The circulation of ions across biological membranes, especially protons and sodium ions, is one of the fundamental processes in cellular energetics. Transport systems pump ions across membranes at the expense of some energy source, establishing an ion gradient whose electrochemical potential represents stored energy. Return of that ion across the membrane is mediated by a second transport system which links the downhill flux of the ion to the performance of some useful work (4). Mitchell's chemiosmotic hypothesis (13, 14) proposed that electron transport and phosphorylation are not chemically linked, but are coupled by a transmembrane current of protons or proton motive force (PMF). The electron transport chain is a metabolic pathway arranged within and across the membrane to translocate protons across it. Since the membrane has a low conductance for protons and ions, a gradient of pH (ApH) and of membrane potential (A/@) will develop across the membrane to form the total PMF (AP) as given by the equation AP = A4 - ZApH (1) where Z equals 2.3 (RTIF) and R, T, and F have their usual meanings. The free energy released by the electron transport chain is transformed into and stored as the electrochemical potential of protons. Finally, the ATP synthase is a second proton-translocating pathway, which utilizes that proton potential to drive the phosphorylation of ATP. However, in 1979, Michels et al. (12) proposed another mechanism for the generation of PMF by organic acid-producing bacteria such as lactococci, streptococci, lactobacilli, clostridia, and Esch-

erichia coli. In this energy-recycling model, metabolic end products and protons are coexcreted via specific symport proteins in the cytoplasmic membrane. As result of this proton translocation, a PMF is generated which can contribute to the overall production of metabolic energy (23, 25). Using Lactococcus cremoris, a homofermentative lactateproducing organism, as a model system, they observed that proton excretion coupled to lactate export supplied a significant quantity of metabolic energy to cells (17, 18, 24, 26). Lactobacillus plantarum belongs to homofermentative lactobacilli which convert 1 mol of glucose to 2 mol of lactate (7). However, L. plantarum also forms small amounts of acetate, acetoin, diacetyl, and 2,3-butanedial under certain environmental conditions (6). In previous chemostat studies, we have found that L. plantarum 8014 catabolizes glucose to acetate at the expense of some lactate and acetoin in response to changes in environmental conditions such as acidity (11) and molecular oxygen supply (16), and we identified the enzyme activities responsible for these changes (27-29). The biomass of L. plantarum 8014 increases in aerobic cultures (16, 28) even though it lacks a complete heme-linked electron transport system (9, 20). This probably occurs because one additional ATP is formed per molecule of pyruvate through substrate-level phosphorylation concurrent with the formation of acetate (20). Michels' energy-recycling model has been proven for lactate and postulated for other organic acids, suggesting that acetate excretion may contribute to electrochemical proton gradient (25). Verdoni et al. (31) have recently published indirect evidence that acetate and formate gradients may contribute to the generation of PMF in Pseudomonas mendocina, but they did not quantify the relative contribution of each organic acid. The objective of this study was to use continuous culture to control medium pH and 02 levels so that the

* Corresponding author. t Paper D-10112-1-91 of the New Jersey State Agricultural Exper-

iment Station.

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independent bioenergetic consequences of these variables on L. plantarum 8014 could be determined. We report here the influences of 02 and pH on L. plantarum 8014 biomass, PMF, intracellular ATP, lactate, and acetate concentrations, and H+/lactate and H+/acetate stoichiometries. The results demonstrate that the extrusion of protons linked to lactate and acetate production played an important role in the cellular energy economy of both transient and steady-state L. plantarum 8014 cells. MATERIALS AND METHODS Organism and continuous culture conditions. L. plantarum ATCC 8014 was maintained as previously described (15) and grown in the modified (11) medium of Craig and Snell (3) designated CST-YNB. Glucose was added to the medium at a final concentration of 25 mM. The chemostat (Mouse; Queue Systems, Parkersburg, W. Va.) was operated as previously described (11) with a 1,000-ml working volume at 35°C, pH 5.5 or 7.5, 250 rpm, and a flow rate of 200 ml h-1 to give a dilution rate of 0.20 h-1. Anaerobiosis was maintained by a continuous nitrogen overlay. The aerobic condition was maintained by sparging the vessel with air at a rate of 1.0 liter min-' (1.84 mmol liter-' min-') as previously described (16). The culture pH was controlled automatically by the addition of 0.5 N HCl or 0.5 N NaOH. To inoculate the culture, 250 ml of an overnight culture were centrifuged, resuspended in 10 ml of 0.9% saline, injected via syringe into the chemostat, and grown as a batch culture until midexponential phase, when continuous feeding of fresh medium was initiated at a dilution rate of 0.20 h-1. Steady state was assumed to require five residence times and was confirmed by the catabolite analysis of three samples taken at least 4 h apart. If the values varied by more than 10%, the chemostat was given additional time to reach steady state. After the first set of steady-state data was obtained, the chemostat was returned to anaerobic steady state at pH 5.5, ard the whole experiment was repeated. Samples were drawn at appropriate intervals during the transient and at steady state. Measurement of ApH and A*. The membrane potential (AI) and proton gradient (ApH) were determined by the distribution of [3H]tetraphenylphosphonium ion (TPP+) and [14C]salicylic acid or [14C]methylamine, respectively, as described by Rottenberg (19). Corrections for nonspecific label binding were based on the extent of label accumulation in control cells treated with 5% butanol and/or 1 ,uM nigeracin for 60 min. Cell suspensions (1 ml) withdrawn from the chemostat were immediately dispensed into microfuge tubes containing 5 ,uM (0.375 RCi) ["4C]salicylic acid or 8 ,uM (0.1 ,uCi) [3H] TPP. After 1 min of incubation, 0.5 ml of silicone oil (density, 1.02 g/ml) was layered on top of the suspension. The cells and supernatant were separated by centrifugation through the silicone oil at 13,000 x g for 3 min. Samples (100 RI) of the supernatant fluid were removed for the measurement of radioactivities. The aqueous layers and most of the silicone oil were then removed by pipette, and the bottoms of the tubes containing the cell pellets were cut off so that they fell directly into scintillation vials containing 5 ml of scintillation fluid (Hydrofluor; National Diagnostics, Highland Park, N.J.). Radioactivity was assayed by using a Beckman LS1801 scintillation counter equipped with a duallabel counting program. The intracellular volume of L. plantarum cells was determined by using 5 ,uCi of 3H20 for the total aqueous space

and subtracting the space occupied by 3 ,uCi of ['4C]inulin. The calculated intracellular value was 2.57 RI/mg (dry weight) of cells. Measurement of intracellular ATP, lactate, and acetate concentrations. The intracellular lactate and acetate concentrations were detected by using 1.0-ml aliquots taken directly from the chemostat and transferred to a microfuge tube containing 0.5 ml of silicone oil (density, 1.05 g/ml) on top of 0.2 ml of 22% (wt/wt) perchloric acid (PCA; density, 1.14 g/mg). The cells were separated from the external medium by centrifugation for 2 min at room temperature and 14,000 x g in a microfuge. In PCA, rapid disruption of the cells inhibits further metabolism and releases the intracellular metabolites (24). The supernatants and the PCA fraction were neutralized by 3 M KOH to pH 7.0, and the lactate and acetate concentrations were determined enzymatically (2, 10). From the amounts of intracellular and extracellular water and the lactate and acetate concentrations in the supernatants, the internal lactate and acetate concentrations were calculated. The corresponding gradients were calculated by the Nernst equation: AUOA = 2.3RT/F(log[OA]i,/[OA]ex) (2) with 2.3RTIF = 60.44 mV, where OAi and OA,X are intracellular and extracellular organic acids. The ATP content in the cells was estimated by the bioluminescence method (22) using an LKB luminometer. Chemicals. The yeast extract was purchased from Difco Laboratories (Detroit, Mich.). TPP+ (23 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, United Kingdom. 3H20 (250 i,Ci/ml), [14C]salicylic acid (58.2 mCi/ mmol), [14C]inulin (2.00 mCi/g), and [14C]methylamine (48.9 mCi/mmol) were purchased from Du Pont Co., Wilmington, Del. Silicone oil (density, 1.05 g/ml) was purchased from Aldrich Chemical Co., Inc. All other chemicals were purchased from Sigma Chemical Co.

RESULTS Effect of oxygen on the intracellular pH, ATP, lactate, acetate, and PMF. The PMF of L. plantarum 8014 increased about 20% and the intracellular ATP concentration increased 13% immediately after the culture was shifted from anaerobic to aerobic conditions. These two energy parameters dropped as biomass accumulated and then increased again at 2 h (ATP) or 3 h (PMF) after the shift to aerobic conditions (Fig. 1). However, when the culture reached steady state in the aerobic chemostat, both values were lower than during anaerobic steady state (Table 1). The changes in intracellular ATP concentrations paralleled the changes of PMF. The intracellular concentrations of lactate and acetate, which export protons that contribute to the proton gradient, also tended to change in the same fashion as the total PMF (Fig. 1). The variable proton

TABLE 1. Steady-state culture parameters for L. plantarum chemostat cultures (D = 0.2 h-1, pH 5.5, 25 mM glucose) under aerobic and anaerobic conditions Condition

Anaerobic Aerobic

Alp PMF Intracel lular pH mV) (mV) 6.6 6.3

53 31

112 78

Intracel-

n

(MM)

Lactate

3.8 1.6

0.8 0.8

value Acetate

0.8 1.1

BIOENERGETIC CHANGES

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E

4413

2.50

2~~~~~~~~~~~~~~~~~~~~~~ 0.0

E 0

0.5 '0

1.0

0.50F 0

0

1

2

3

4

5

6

7

8

9

Time (hours) FIG. 1. Transient influence of oxygen (transfer rate, 1.84 mmol liter-' min-') on biomass (Aw), intracellular ATP, lactate, and acetate, and PMF by L. plantarum 8014 grown in an anaerobic chemostat at pH 5.5, D = 0.2 h-1, 25 mM glucose.

stoichiometry (n) of the lactate and acetate carriers during the aerobic transient state (Fig. 2) was calculated according to equation 3 from the data of Fig. 1 (24): n = (A* - AUOA)/AP (3) The H+/lactate excretion stoichiometry varied from 0.8 at anaerobic steady state to 1.2 at 2 h after the shift to aerobic conditions. The H+/acetate stoichiometry changed from 0.8 during anaerobic steady state to a maximum of 1.6 at 2 h after the shift to aerobic conditions (Fig. 2). After cultures achieved aerobic steady state, the n value for lactate excre-

tion decreased to the same value as under anaerobic conditions; however, the n value for acetate excretion remained elevated (Table 1). At each steady state, the intracellular pH, ATP, and A4 were measured; the PMF was calculated by equation 1. L. plantarum 8014 had higher PMF values as well as higher intracellular ATP concentrations in anaerobic steady state than in aerobic steady state (Table 1). Effect of chemostat pH value on intracellular pH, intracellular ATP, lactate, and acetate concentrations, and PMF. After the culture achieved anaerobic steady state at pH 5.5,

1.8

6

1.

A

Time (hours)

FIG. 2. Transient influence of oxygen on H+/lactate and H+/acetate stoichiometries in L. plantarum 8014 cells. indicated time from the data shown in Fig. 1 by using equation 3.

n was

calculated at each

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Acetate mM xl o

-f

E E E 0

0. c ~~

~ ~ ~ ~ ~ ~~Tm ohors

-0.5

0.5

1.5

2.5

3.5

4.5

5.5

6.5

7.5

8.5

Tim. (hours) FIG. 3. Transient influence of medium pH change from 5.5 (at time -0.5) to 7.5 (at time zero) on biomass (Aw), intracellular ATP, lactate, and acetate, and PMF by L. plantarum 8014 grown in an anaerobic chemostat at D = 0.2 h-1, 25 mM glucose.

the medium pH was shifted from 5.5 to 7.5 by rapid addition of 1.0 N NaOH. The intracellular ATP concentrations increased 20% immediately, then decreased quickly 45 min after the shift to pH 7.5, and later decreased again 3 h postshift (Fig. 3). The intracellular ATP, lactate, and acetate concentrations also followed an oscillatory pattern. The PMF of the cells decreased 50% when the pH was shifted to 7.5 as a result of the depletion of the transmembrane proton gradient, but the biomass did not change dramatically even 9 h after the shift to pH 7.5. The changes in the intracellular lactate and acetate concentrations were consistent with the changes in intracellular ATP concentrations.

During the transient state after the shift to pH 7.5, the H+/lactate and H+/acetate n values (Fig. 4) were calculated from the data of Fig. 3 by using equation 3. The H+/Ilactate n value varied from 0.7 to the maximum of 2.1 and was maintained at 1.8 when the cells achieved pH 7.5 steady state. The H+/acetate n value changed from 0.9 to the maximum of 2.8 but decreased to 1.9 at the pH 7.5 steady state (Fig. 4 and Table 2). The intracellular pH increased from 6.6 to 7.3, and A*i changed from 42 mV to 63 mV at the steady state of pH 7.5. However, the PMF decreased from 106 mV to 55 mV and the intracellular ATP decreased from 3.6 mM to 1.1 mM (Table 2).

3,

CS

U

o +

o0.5

.

-.--i-

0.5

1.5

2.5

3.5

4.5

5.5

6.5

7.5

8.5

Time (hours) FIG. 4. Transient influence of medium pH change from 5.5 (at time -0.5) to 7.5 (at time zero) on H+/lactate and H+/acetate stoichiometries in L. plantarum 8014 cells. n was calculated at each indicated time from the data shown in Fig. 3 by using equation 3.

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BIOENERGETIC CHANGES

TABLE 2. Steady-state culture parameters for anaerobic L. plantarum chemostat cultures (D = 0.2 h-1, 25 mM glucose) under acidic and alkaline conditions Chemostat pH

Intracellular pH

5.5 7.5

6.6 7.3

A* PMF

(mV) (mV)

42 63

Intracel(mM)

Lactate

Acetate

3.6 1.1

0.7 1.8

0.9 1.9

106 55

n value

DISCUSSION It is generally accepted that bacteria establish and maintain an electrochemical gradient of protons (AUH+) across their cytoplasmic membranes. The proton gradient is interconvertible with ATP by means of the H+-translocating ATPase (ATP synthase BFoF1) (4, 8). Strictly fermentative lactic acid bacteria lack a complete electron transfer system that functions as a proton pump. In these bacteria, the ATPase functions in the direction of hydrolysis, and the H+ excretion required to generate PMF causes less ATP to be available for biosynthetic purposes (5). To avoid this drain of ATP, these organisms have developed transport systems that mediate the efflux of metabolic end products in symport with protons. This generates PMF if net charge is translocated (12). However, direct experimental proof of this energy-recycling model has previously limited to lactate excretion in homofermentative lactic acid bacteria such as L. cremoris (17, 18, 24, 26) and to the vesicles of E. coli (23). ATPase inhibitor studies suggested that excretion of mixed acids may be associated with P. mendocina growth (31). L. plantarum 8014 produces acetate in addition to lactate in aerobic and alkaline environments (11, 16) as a result of changes in the corresponding catabolic enzyme activities (27-29). In this study, we observed that L. plantarum 8014 increased PMF and intracellular ATP immediately after the anaerobic chemostat was shifted to aerobic conditions (Fig. 1) in which NADH oxidase is induced to allow NAD regeneration (27, 28). The increased intracellular ATP at the beginning of shift was probably due to the fact that one additional ATP was formed with the formation of acetate. Lactic acid bacteria usually maintain a constant intracellular pH (8); therefore, the subsequent decrease in intracellular ATP may have been caused by its use for biosynthesis or exporting of protons produced by end products to maintain intracellular pH homeostasis. In aerobic conditions, the changes in PMF occurred slightly after, but consistent with, the changes of intracellular ATP as well as the changes of H+/lactate and H+/acetate excretion stoichiometries. These results suggest that protons excreted with end products or by ATPase-mediated ATP hydrolysis could generate a PMF in L. plantarum 8014. This also would explain the increased PMF at the beginning and the third hour after the shift to aerobic conditions (Fig. 1 and 2). L. plantarum 8014 produced more acidic compounds with correspondingly higher intracellular levels of organic acids, causing the decrease of intracellular pH at aerobic steady state (Table 1). The excretion of protons by the ATPase, causing intracellular ATP to decrease, would be coupled with the electrogenic uptake of potassium ions (1), explaining the decrease in membrane potential (inside negative) at aerobic steady state. In alkaline conditions, L. plantarum 8014 diverts some of its lactate and acetoin to the production of acetate. The export of more protons in conjunction with lactate and acetate would reduce the need of ATP for ATPase-mediated

4415

proton excretion. This and the one extra ATP generated by acetate kinase when acetate is produced (28, 29) would explain the increased ATP levels during the transient state at pH 7.5 under anaerobic conditions (Fig. 3). Because L. plantarum 8014 produced more acidic compounds at pH 7.5, the intracellular pH was lower than the extracellular pH, minimizing the ApH component of PMF. However, Au increased 50%, partially compensating for the depletion of ApH. The 70% decrease in intracellular ATP might have been caused by export of protons to neutralize extracellular pH and maintain intracellular pH. Alternately, the decrease may have been from the hydrolysis of ATP used to drive the uptake of essential nutrients from the medium (21, 30). The steady-state n values reported here increased with increasing pH and are similar to the values of 0.9 at pH 5.5 to 1.9 at pH 7.0 reported for L. cremoris (24). H+/acetate excretion stoichiometries as high as 2.8 during the transient may be of special significance to organisms which grow in ecological niches that are usually nutritionally depleted but subject to period fluxes of high nutrient density. The fact that n values for acetate are higher than those for lactate suggests that producing acetate at the expense of lactate not only results in an additional ATP from substrate-level phosphorylation but, at n = 3, would spare an additional ATP that would otherwise be used to maintain ApH (24). In this study, we have demonstrated the bioenergetic consequences of acetate production by L. plantarum 8014 produced in aerobic and alkaline conditions. The H+/lactate and H+/acetate excretion stoichiometries increased in both the transient states as well as in the pH 7.5 steady state. This finding suggests that L. plantarum 8014 coexcreted more protons with organic acid end products to maintain intracellular pH homeostasis and augment the cellular energy economy. In addition, we report for the first time that the H+/acetate stoichiometry was higher than the H+/lactate stoichiometry and that both H+/lactate and H+/acetate stoichiometries were higher in alkaline than in acidic and aerobic conditions, thus expanding the utility of Michels' energyrecycling model. ACKNOWLEDGMENTS This study was supported by state appropriations and U. S. Hatch funds. REFERENCES 1. Abee, T., K. J. Hellingwerf, and W. N. Konings. 1988. Effects of potassium ions on proton motive force in Rhodobacter sphaeroides. J. Bacteriol. 170:5647-5653. 2. Bergmeyer, H. U. 1983. Determination of metabolite concentration by endpoint methods, p. 163-175. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis, 3rd ed., vol. 1. Verlag Chemie, Weinheim, Germany, and Academic Press, Inc., New York. 3. Craig, J. A., and E. E. Snell. 1951. The comparative activities of pathethine, pantothenic acid and coenzyme A for various microorganisms. J. Bacteriol. 61:238-291. 4. Harold, F. M. 1986. The vital force: a study of bioenergetics. W. H. Freeman & Co., New York. 5. Hellingwerf, K. J., and W. N. Konings. 1985. The energy flow in bacteria: the main free energy intermediates and their regulatory role. Adv. Microb. Physiol. 26:125-154. 6. Kandler, 0. 1983. Carbohydrate metabolism in lactic acid bacteria. Antonie Van Leeuwenhoek J. Microbiol. Serol. 49: 209-224. 7. Kandler, O., and N. Weiss. 1984. Regular, nonsporing Grampositive rods, p. 1208-1260. In P.H.A. Sneath (ed.), Bergey's manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore.

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