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13C nuclear magnetic resonance analysis of glucose and citrate end products in an ldhL-ldhD double-knockout strain of Lactobacillus plantarum. T Ferain, A N Schanck and J Delcour J. Bacteriol. 1996, 178(24):7311.
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JOURNAL OF BACTERIOLOGY, Dec. 1996, p. 7311–7315 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 178, No. 24
13
C Nuclear Magnetic Resonance Analysis of Glucose and Citrate End Products in an ldhL-ldhD Double-Knockout Strain of Lactobacillus plantarum T. FERAIN,1 A. N. SCHANCK,2
AND
J. DELCOUR1*
Laboratoire de Ge´ne´tique Mole´culaire, Unite´ de Ge´ne´tique,1 and Laboratoire de Chimie Physique et de Cristallographie,2 Universite´ Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
We have examined the metabolic consequences of knocking out the two ldh genes in Lactobacillus plantarum using 13C nuclear magnetic resonance. Unlike its wild-type isogenic progenitor, which produced lactate as the major metabolite under all conditions tested, ldh null strain TF103 mainly produced acetoin. A variety of secondary end products were also found, including organic acids (acetate, succinate, pyruvate, and lactate), ethanol, 2,3-butanediol, and mannitol. Lactobacillus plantarum is a lactic acid bacterium that ferments glucose via the homofermentative pathway. Glucose is first converted to pyruvate through the Embden-MeyerhofParnas pathway, and most of the pyruvate is further reduced to D- and L-lactate by stereospecific NAD-dependent lactate dehydrogenases (LDHs). In addition to lactate, L. plantarum (and other lactic acid bacteria) can convert pyruvate to a variety of secondary end products (Fig. 1) whose distribution depends on strains and culture parameters. For instance, production of acetate from pyruvate via acetylphosphate has been largely reported for L. plantarum grown on glucose under aerobic conditions (14, 16, 33, 41, 42). The enzyme responsible for this conversion is pyruvate oxidase (15, 34–36, 41). Acetate production from glucose has also been observed in L. plantarum under anaerobiosis at neutral or alkaline pH (27, 33, 42), as well as in the presence of an external electron acceptor, such as citrate (25). Likewise, production of ethanol from glucose has also been reported for both aerobic and anaerobic cultures of L. plantarum (7, 28, 33). Ethanol can be formed from pyruvate via acetyl coenzyme A and acetaldehyde, with actyl coenzyme A resulting from the anaerobic pyruvate formate-lyase pathway. The presence of this pathway in a few strains of L. plantarum has been suggested (12, 25), but pyruvate formatelyase activity has not been demonstrated. Ethanol is also produced from external electron acceptors, such as citrate and acetate, during anaerobic catabolism of mannitol in L. plantarum (28). Moreover, during glucose fermentation, L. plantarum frequently produces C4 compounds from pyruvate via a-acetolactate. The formation of these metabolites (diacetyl, acetoin, and 2,3-butanediol) is enhanced by the addition of exogenous pyruvate (9, 16, 27, 30, 31) and is also stimulated by low pH values (30, 41). Citrate is responsible for a large variation of fermentation profiles in lactic acid bacteria (17). Its catabolism usually generates additional pyruvate without reduction of the NAD cofactor, and pyruvate is further channeled towards the formation of the above-mentioned end products. Another pathway of citrate dissipation in L. plantarum and some other lactoba-
cilli is the production of succinate via L-malate and fumarate (20, 21, 32). We have previously described the physiological impact of knocking out the two ldh genes in L. plantarum (11). In this report, we examine the spectrum of end products from glucose metabolism in the ldh null mutant (strain TF103) in comparison with its wild-type isogenic progenitor (strain NCIMB8826; National Collection of Industrial and Marine Bacteria, Torry Research Station Aberdeen, Scotland). Cells were grown under aerobiosis or anaerobiosis and in the presence or absence of citrate in addition to glucose. End products were identified by 13C nuclear magnetic resonance (Fig. 2; Table 1) according to the method of Chaumont et al (6). Glucose and glucose-citrate cometabolism in the wild-type strain NCIMB8826. Glucose catabolism was first investigated for the wild-type strain NCIMB8826. This strain was grown in FT80 medium (3), which was supplemented with 10 mM citrate for glucose-citrate cometabolism studies. For 13C nuclear magnetic resonance analysis (Fig. 2), cells were resuspended in 70 mM potassium–sodium phosphate buffer (pH 5.5) supplemented with 50 mM glucose, together with 10 mM citrate, for cometabolism studies. During aerobic incubation, glucose was mainly converted to lactate and to a lesser extent to acetate. Under anaerobic conditions, glucose was exclusively converted to lactate (Table 1). These observations are in agreement with the well-known oxygen-dependent formation of acetate via pyruvate oxidase (15, 34, 36, 41). Cometabolism of citrate and glucose led to acetate production under anaerobiosis and enhanced acetate formation in the presence of oxygen (Table 1). Acetate is produced along with oxalacetate during the first step of citrate degradation (Fig. 1). Oxalacetate is usually decarboxylated to pyruvate, which is further dissipated into by-products, such as C4 compounds (9, 30, 31). However, these metabolites were not observed with the wild-type strain in our experiments. Aerobic glucose metabolism in the LDH-deficient strain TF103. Glucose metabolism in the mutant was carried out as described above for the wild-type strain but only under aerobic conditions (Table 1), since TF103 is unable to grow on glucose alone in the absence of oxygen. Acetoin was the main end product. The synthesis of this compound is known to require a high concentration of intracellular pyruvate (39, 40), which is most likely the case for our ldh null mutant. Ethanol was also produced by TF103 during aerobic catab-
* Corresponding author. Mailing address: Laboratoire de Ge´ne´tique Mole´culaire, Unite´ de Ge´ne´tique, Universite´ Catholique de Louvain, Place Croix du Sud, 5 (bte 6), B-1348 Louvain-la-Neuve, Belgium. Phone: (32)(10)473484. Fax: (32)(10)473109. Electronic mail address: [email protected]. 7311
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Received 11 April 1996/Accepted 4 October 1996
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olism of glucose. We hypothesize that it does not result from pyruvate formate-lyase activity, since this pathway also leads to formate accumulation and has been reported for other species only under strict anaerobic conditions (13). Ethanol production from external electron acceptors (acetate, citrate, and oxalacetate) during anaerobic catabolism of mannitol in L. plantarum has been previously described (28, 37). According to these studies, ethanol derives from acetate via acetyl phosphate, acetyl coenzyme A, and acetaldehyde. All the enzymes involved in this pathway were shown to be constitutive in wildtype L. plantarum (7). Aerobic ethanol production in the LDHdefective strain therefore probably stemmed from acetyl phosphate arising from pyruvate oxidase activity. Likewise, part of this intermediate was also converted to acetate, since this metabolite was also produced under these conditions. Low concentrations of mannitol were also detected. As mentioned above, L. plantarum is able to use this hexose as a carbon source (37). This pathway, which involves NAD1 reduction, might be reversed in our strain. Under this condition,
fructose 6-phosphate formed from glucose could be converted to mannitol (Fig. 1), lowering the NAD1 debt. Small amounts of mannitol have been described as end products of Streptococcus mutans in the presence of large concentrations of sucrose or glucose (26). The presence of mannitol-1-phosphate dehydrogenase has been demonstrated for both TF103 and NCIMB8826 extracts (Table 2). Low concentrations of lactate were also measured. Both D and L isomers were observed with a large excess of L-lactate (80%). However, no LDH activity was present in the mutant strain (Table 2). Other NAD-dependent dehydrogenases able to reduce a-keto acids to the corresponding a-hydroxy acids in vitro in lactic acid bacteria have been previously described (19). Some of these stereospecific enzymes (known as hydroxyisocaproate dehydrogenases) are able to weakly reduce pyruvate to lactate in vitro (1, 23, 24). It was reported that during anaerobic mannitol fermentation, L. plantarum was able to use several a-keto acids as electron acceptors for NAD1 regeneration (a-ketobutyric acid, a-ketovaleric acid, and a-ketocapry-
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FIG. 1. Glucose and citrate degradation pathways proposed for the L. plantarum wild-type strain (NCIMB8826) and LDH-defective strain (TF103). [1], LDH; [2], pyruvate formate-lyase; [3], acetaldehyde dehydrogenase; [4], alcohol dehydrogenase; [5], phosphotransacetylase; [6], acetate kinase; [7], pyruvate oxidase; [8], pyruvate decarboxylase; [9], a-acetolactate synthase; [10], a-acetolactate decarboxylase; [11], 2,3-butanediol dehydrogenase; [12], nonenzymatic decarboxylation; [13], pyruvate carboxylase; [14], malate dehydrogenase; [15], fumarase; [16], fumarate reductase; [17], malolactic enzyme; [18], citrate lyase; [19], oxalacetate decarboxylase; [20], mannitol-1-phosphate dehydrogenase; [21], mannitol-1-phosphatase–enzyme IIMtl. CoA, coenzyme A; 2 e2, electrons transferred to an unknown acceptor; TPP, thiamine PPi.
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FIG. 2. 13C nuclear magnetic resonance spectra of end products from cell suspensions of wild-type L. plantarum NCIMB8826 (A) and LDH-defective L. plantarum TF103 (B) under the following conditions: aerobiosis and 50 mM glucose (a); aerobiosis and 50 mM glucose plus 10 mM citrate (b); anaerobiosis and 50 mM glucose (c); and anaerobiosis and 50 mM glucose plus 10 mM citrate (d). ACN, acetoin; ACT, acetate; BUT, 2,3-butanediol; CIT, citrate; black triangles, resonance of residual glucose; ETH, ethanol; LAC, lactate; MAN, mannitol; PYR, pyruvate; SUC, succinate. Spectra are presented from 15 to 75 ppm. Carbon atoms of the carbonyl groups were also detected, but under our experimental conditions with a 6-s repetition time, those signals were not quantitative.
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TABLE 1. End producta concentrations from glucose and glucose-citrate cometabolism by cell suspensions of wild-type (NCIMB8826) and LDH-defective (TF103) strains of L. plantarum Concn (mM) of end product: Incubation
Strain
Carbon source
Aerobic
a
Citrate
Lactate
Acetate
8 6
4
72 77
6 12
2 14
1 3
11 3
75 82
6
18
4
2
7
TABLE 2. Enzymatic activities detected in cell extractsa from the TF103 and NCIMB8826 strains of L. plantarum Enzyme
Sp act (U/mg of proteinb) of strain: TF103
Pyruvate carboxylase Malate dehydrogenase Malolactic enzyme Mannitol-1 phosphate dehydrogenase LDH 10.
Reference(s) for assays
NCIMB8826 23
6 3 10 173 3 1023
5 3 1023 NAc
25 3 1023 23 3 1023
NA 29 3 1023
NDd
14
29 5 2, 22 4 10
Cell extracts were prepared according to the method described in reference
One unit of activity corresponds to the conversion of 1 mmol of substrate per min. c NA, not applicable because of the high level of LDH activity and large amount of lactate in the wild-type strain, which interfere with malate dehydrogenase and malolactic enzyme assays, respectively. d ND, not detected. b
Acetoin
Mannitol
8 4
35 30
4 ,1
3
20
6
2,3Butanediol
2
Succinate
Pyruvate
2 3
3
2
The production of CO2 and volatile compounds was not measured during this study.
lic acid) (28), and the authors hypothesized the involvement of hydroxyisocaproate dehydrogenase activities. However, we did not detect any hydroxyisocaproate dehydrogenase activity in TF103 cell extracts using several a-keto acids as substrates. As to L-lactate production, one possible pathway could be oxalacetate reduction to L-malate followed by malolactic conversion to L-lactate. The two enzymes responsible for these reactions are malate dehydrogenase and the malolactic enzyme (Fig. 1). They have been reported for L. plantarum and other lactobacilli (2, 32) and have also been detected in TF103 (Table 2). Oxalacetate itself may stem from glycolytic phosphoenolpyruvate or pyruvate via the corresponding carboxylases. These enzymatic activities have been described for several bacterial species (13), but to our knowledge, they have not been reported for lactic acid bacteria. Yet, TF103 extracts showed pyruvate carboxylase activity (Table 2), which is also present in the wild-type strain, but no phosphoenolpyruvate carboxylase activity. The observation of small amounts of succinate (Table 1), another metabolite derived from oxalacetate via malate (Fig. 1), supports this model. Aerobic and anaerobic glucose-citrate cometabolism in LDH-deficient strain TF103. A typical anaerobic pathway frequently observed in lactic acid bacteria is pyruvate formatelyase, which leads to formate accumulation. However, the lack
a
Ethanol
of this compound in buffered cell suspensions from anaerobically grown cultures suggests the absence of this pathway. It must be mentioned that the preparation of cell suspensions included manipulations in the presence of oxygen. Therefore, pyruvate formate-lyase (if any) is likely to have been inactivated, since the enzyme is reported to be highly sensitive to oxygen (8). Most of the end products detected during aerobic and anaerobic glucose-citrate cometabolism were the same as those derived from glucose alone (Table 1). Acetoin remained the major metabolite. We also identified trace amounts of 2,3butanediol resulting from acetoin reduction. Lactate production was largely increased, and all the additional lactate was in the form of the L isomer. This fits the above-described hypothesis of L-lactate production through malolactic fermentation; under these conditions, oxalacetate would arise from citrate (Fig. 1). On the other hand, levels of ethanol and mannitol production were lower in the presence of citrate, which could reflect NAD1 regeneration via other pathways owing to citrate utilization as the external electron acceptor. Small amounts of pyruvate were also detected. Wild-type Lactococcus lactis and Leuconostoc spp. have been reported to excrete pyruvate under conditions in which C4 compounds are formed, i.e., during citrate fermentation (18, 38), and the presence of these compounds has been correlated with an internal accumulation of high concentrations of pyruvate, which very likely occurs in the LDH-defective strain TF103 of L. plantarum. This research was carried out within the framework of the European Community Research Programme ECLAIR with financial contributions from the Commission (contract AGRE-CT90-0041) and from the General Directorate for Research and Technology of the Walloon Region (contract 1580). T. Ferain holds an I.R.S.I.A. fellowship. We are indebted to E. Van Schaftingen and M. Veiga da Cunha for helpful discussions and comments during the course of this work. REFERENCES 1. Bernard, N., K. Johnsen, T. Ferain, D. Garmyn, P. Hols, and J. J. Holbrook. 1994. NAD1-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization. Eur. J. Biochem. 224:439–446. 2. Caspritz, G., and F. Radler. 1983. Malolactic enzyme of Lactobacillus plantarum. J. Biol. Chem. 258:4907–4910. 3. Cavin, J. F., H. Prevost, J. Lin, P. Schmitt, and C. Divies. 1989. Medium for screening Leuconostoc oenos strains defective in malolactic fermentation. Appl. Environ. Microbiol. 55:751–753. 4. Chakravorty, M. 1964. Metabolism of mannitol and induction of mannitol 1-phosphate dehydrogenase in Lactobacillus plantarum. J. Bacteriol. 87: 1246–1248.
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NCIMB8826 Glucose NCIMB8826 Glucose plus citrate TF103 Glucose TF103 Glucose plus citrate Anaerobic NCIMB8826 Glucose NCIMB8826 Glucose plus citrate TF103 Glucose plus citrate
Glucose
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5. Charnock, C., U. H. Refseth, and R. Siervåg. 1992. Malate dehydrogenase from Chlorobium vibrioforme, Chlorobium tepidum, and Heliobacterium gestii: purification, characterization, and investigation of dinucleotide binding by dehydrogenases by use of empirical methods of protein sequence analysis. J. Bacteriol. 174:1307–1313. 6. Chaumont, F., A. N. Schanck, J. J. Blum, and F. R. Opperdoes. 1994. Aerobic and anaerobic glucose metabolism of Phytomonas sp. isolated from Euphorbia characias. Mol. Biochem. Parasitol. 67:321–331. 7. Chen, K.-H., and R. F. McFeeters. 1986. Utilization of electron acceptors in anaerobic metabolism by Lactobacillus plantarum. Enzymes and intermediates in the utilization of citrate. Food Microbiol. 3:83–92. 8. Condon, S. 1987. Responses of lactic acid bacteria to oxygen. FEMS Microbiol. Rev. 46:269–280. 9. El-Gendy, S. M., H. Abdel-Galil, Y. Shahin, and F. Z. Hegazi. 1983. Acetoin and diacetyl production by Lactobacillus plantarum able to use citrate. J. Food Prot. 46:503–505. 10. Ferain, T., D. Garmyn, N. Bernard, P. Hols, and J. Delcour. 1994. Lactobacillus plantarum ldhL gene: overexpression and deletion. J. Bacteriol. 176: 596–601. 11. Ferain, T., J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour. 1996. Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum. J. Bacteriol. 178:5431–5437. 12. Fryer, T. F. 1970. Utilization of citrate by lactobacilli isolated from dairy products. J. Dairy Res. 37:9–15. 13. Gottschalk, G. (ed.) 1988. Bacterial metabolism, 2nd ed. Springer-Verlag, New York. 14. Go¨tz, F., and B. Sedewitz. 1991. Physiological role of pyruvate oxidase in the aerobic metabolism in Lactobacillus plantarum, p. 286–293. In H. Bisswanger and J. Ullrich (ed.), Biochemistry and physiology of thiamin diphosphate enzymes. Verlag Chemie, Weinheim, Germany. 15. Go¨tz, F., B. Sedewitz, and E. F. Elstner. 1980. Oxygen utilisation by Lactobacillus plantarum. I. Oxygen consuming reactions. Arch. Microbiol. 125: 209–214. 16. Hickey, M. W., A. J. Hillier, and G. R. Jago. 1983. Metabolism of pyruvate and citrate in lactobacilli. Aust. J. Biol. Sci. 36:487–496. 17. Hugenholtz, J. 1993. Citrate metabolism in lactic acid bacteria. FEMS Microbiol. Rev. 12:165–178. 18. Hugenholtz, J., and J. C. Starrenburg. 1992. Diacetyl production by different strains of Lactococcus lactis subsp. lactis var. diacetylactis. Appl. Microbiol. Biotechnol. 38:17–22. 19. Hummel, W., and M.-R. Kula. 1989. Dehydrogenases for the synthesis of chiral compounds. Eur. J. Biochem. 184:1–13. 20. Kaneuchi, C., M. Seki, and K. Komagata. 1988. Production of succinic acid from citric acid and related acids by Lactobacillus strains. Appl. Environ. Microbiol. 54:3053–3056. 21. Kennes, C., H. C. Dubourguier, G. Albagnac, and E.-J. Nyns. 1991. Citrate metabolism by Lactobacillus plantarum isolated from orange juice. J. Appl. Bacteriol. 70:380–384. 22. Labarre, C., J. Guzzo, J.-F. Cavin, and C. Divie`s. 1996. Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos. Appl. Environ. Microbiol. 62:1274–1282. 23. Lerch, H.-P., H. Blo¨cker, H. Kallwas, J. Hoppe, H. Tsai, and J. Collins. 1989. Cloning, sequencing and expression in Escherichia coli of the D-2-hydroxyisocaproate dehydrogenase gene of Lactobacillus casei. Gene 78:47–57. 24. Lerch, H.-P., R. Frank, and J. Collins. 1989. Cloning, sequencing and expression of the L-2-hydroxyisocaproate dehydrogenase-encoding gene of
NOTES
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JOURNAL OF BACTERIOLOGY, Dec. 1996, p. 7311–7315 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 178, No. 24
13
C Nuclear Magnetic Resonance Analysis of Glucose and Citrate End Products in an ldhL-ldhD Double-Knockout Strain of Lactobacillus plantarum T. FERAIN,1 A. N. SCHANCK,2
AND
J. DELCOUR1*
Laboratoire de Ge´ne´tique Mole´culaire, Unite´ de Ge´ne´tique,1 and Laboratoire de Chimie Physique et de Cristallographie,2 Universite´ Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
We have examined the metabolic consequences of knocking out the two ldh genes in Lactobacillus plantarum using 13C nuclear magnetic resonance. Unlike its wild-type isogenic progenitor, which produced lactate as the major metabolite under all conditions tested, ldh null strain TF103 mainly produced acetoin. A variety of secondary end products were also found, including organic acids (acetate, succinate, pyruvate, and lactate), ethanol, 2,3-butanediol, and mannitol. Lactobacillus plantarum is a lactic acid bacterium that ferments glucose via the homofermentative pathway. Glucose is first converted to pyruvate through the Embden-MeyerhofParnas pathway, and most of the pyruvate is further reduced to D- and L-lactate by stereospecific NAD-dependent lactate dehydrogenases (LDHs). In addition to lactate, L. plantarum (and other lactic acid bacteria) can convert pyruvate to a variety of secondary end products (Fig. 1) whose distribution depends on strains and culture parameters. For instance, production of acetate from pyruvate via acetylphosphate has been largely reported for L. plantarum grown on glucose under aerobic conditions (14, 16, 33, 41, 42). The enzyme responsible for this conversion is pyruvate oxidase (15, 34–36, 41). Acetate production from glucose has also been observed in L. plantarum under anaerobiosis at neutral or alkaline pH (27, 33, 42), as well as in the presence of an external electron acceptor, such as citrate (25). Likewise, production of ethanol from glucose has also been reported for both aerobic and anaerobic cultures of L. plantarum (7, 28, 33). Ethanol can be formed from pyruvate via acetyl coenzyme A and acetaldehyde, with actyl coenzyme A resulting from the anaerobic pyruvate formate-lyase pathway. The presence of this pathway in a few strains of L. plantarum has been suggested (12, 25), but pyruvate formatelyase activity has not been demonstrated. Ethanol is also produced from external electron acceptors, such as citrate and acetate, during anaerobic catabolism of mannitol in L. plantarum (28). Moreover, during glucose fermentation, L. plantarum frequently produces C4 compounds from pyruvate via a-acetolactate. The formation of these metabolites (diacetyl, acetoin, and 2,3-butanediol) is enhanced by the addition of exogenous pyruvate (9, 16, 27, 30, 31) and is also stimulated by low pH values (30, 41). Citrate is responsible for a large variation of fermentation profiles in lactic acid bacteria (17). Its catabolism usually generates additional pyruvate without reduction of the NAD cofactor, and pyruvate is further channeled towards the formation of the above-mentioned end products. Another pathway of citrate dissipation in L. plantarum and some other lactoba-
cilli is the production of succinate via L-malate and fumarate (20, 21, 32). We have previously described the physiological impact of knocking out the two ldh genes in L. plantarum (11). In this report, we examine the spectrum of end products from glucose metabolism in the ldh null mutant (strain TF103) in comparison with its wild-type isogenic progenitor (strain NCIMB8826; National Collection of Industrial and Marine Bacteria, Torry Research Station Aberdeen, Scotland). Cells were grown under aerobiosis or anaerobiosis and in the presence or absence of citrate in addition to glucose. End products were identified by 13C nuclear magnetic resonance (Fig. 2; Table 1) according to the method of Chaumont et al (6). Glucose and glucose-citrate cometabolism in the wild-type strain NCIMB8826. Glucose catabolism was first investigated for the wild-type strain NCIMB8826. This strain was grown in FT80 medium (3), which was supplemented with 10 mM citrate for glucose-citrate cometabolism studies. For 13C nuclear magnetic resonance analysis (Fig. 2), cells were resuspended in 70 mM potassium–sodium phosphate buffer (pH 5.5) supplemented with 50 mM glucose, together with 10 mM citrate, for cometabolism studies. During aerobic incubation, glucose was mainly converted to lactate and to a lesser extent to acetate. Under anaerobic conditions, glucose was exclusively converted to lactate (Table 1). These observations are in agreement with the well-known oxygen-dependent formation of acetate via pyruvate oxidase (15, 34, 36, 41). Cometabolism of citrate and glucose led to acetate production under anaerobiosis and enhanced acetate formation in the presence of oxygen (Table 1). Acetate is produced along with oxalacetate during the first step of citrate degradation (Fig. 1). Oxalacetate is usually decarboxylated to pyruvate, which is further dissipated into by-products, such as C4 compounds (9, 30, 31). However, these metabolites were not observed with the wild-type strain in our experiments. Aerobic glucose metabolism in the LDH-deficient strain TF103. Glucose metabolism in the mutant was carried out as described above for the wild-type strain but only under aerobic conditions (Table 1), since TF103 is unable to grow on glucose alone in the absence of oxygen. Acetoin was the main end product. The synthesis of this compound is known to require a high concentration of intracellular pyruvate (39, 40), which is most likely the case for our ldh null mutant. Ethanol was also produced by TF103 during aerobic catab-
* Corresponding author. Mailing address: Laboratoire de Ge´ne´tique Mole´culaire, Unite´ de Ge´ne´tique, Universite´ Catholique de Louvain, Place Croix du Sud, 5 (bte 6), B-1348 Louvain-la-Neuve, Belgium. Phone: (32)(10)473484. Fax: (32)(10)473109. Electronic mail address: [email protected]. 7311
Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV
Received 11 April 1996/Accepted 4 October 1996
7312
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olism of glucose. We hypothesize that it does not result from pyruvate formate-lyase activity, since this pathway also leads to formate accumulation and has been reported for other species only under strict anaerobic conditions (13). Ethanol production from external electron acceptors (acetate, citrate, and oxalacetate) during anaerobic catabolism of mannitol in L. plantarum has been previously described (28, 37). According to these studies, ethanol derives from acetate via acetyl phosphate, acetyl coenzyme A, and acetaldehyde. All the enzymes involved in this pathway were shown to be constitutive in wildtype L. plantarum (7). Aerobic ethanol production in the LDHdefective strain therefore probably stemmed from acetyl phosphate arising from pyruvate oxidase activity. Likewise, part of this intermediate was also converted to acetate, since this metabolite was also produced under these conditions. Low concentrations of mannitol were also detected. As mentioned above, L. plantarum is able to use this hexose as a carbon source (37). This pathway, which involves NAD1 reduction, might be reversed in our strain. Under this condition,
fructose 6-phosphate formed from glucose could be converted to mannitol (Fig. 1), lowering the NAD1 debt. Small amounts of mannitol have been described as end products of Streptococcus mutans in the presence of large concentrations of sucrose or glucose (26). The presence of mannitol-1-phosphate dehydrogenase has been demonstrated for both TF103 and NCIMB8826 extracts (Table 2). Low concentrations of lactate were also measured. Both D and L isomers were observed with a large excess of L-lactate (80%). However, no LDH activity was present in the mutant strain (Table 2). Other NAD-dependent dehydrogenases able to reduce a-keto acids to the corresponding a-hydroxy acids in vitro in lactic acid bacteria have been previously described (19). Some of these stereospecific enzymes (known as hydroxyisocaproate dehydrogenases) are able to weakly reduce pyruvate to lactate in vitro (1, 23, 24). It was reported that during anaerobic mannitol fermentation, L. plantarum was able to use several a-keto acids as electron acceptors for NAD1 regeneration (a-ketobutyric acid, a-ketovaleric acid, and a-ketocapry-
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FIG. 1. Glucose and citrate degradation pathways proposed for the L. plantarum wild-type strain (NCIMB8826) and LDH-defective strain (TF103). [1], LDH; [2], pyruvate formate-lyase; [3], acetaldehyde dehydrogenase; [4], alcohol dehydrogenase; [5], phosphotransacetylase; [6], acetate kinase; [7], pyruvate oxidase; [8], pyruvate decarboxylase; [9], a-acetolactate synthase; [10], a-acetolactate decarboxylase; [11], 2,3-butanediol dehydrogenase; [12], nonenzymatic decarboxylation; [13], pyruvate carboxylase; [14], malate dehydrogenase; [15], fumarase; [16], fumarate reductase; [17], malolactic enzyme; [18], citrate lyase; [19], oxalacetate decarboxylase; [20], mannitol-1-phosphate dehydrogenase; [21], mannitol-1-phosphatase–enzyme IIMtl. CoA, coenzyme A; 2 e2, electrons transferred to an unknown acceptor; TPP, thiamine PPi.
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FIG. 2. 13C nuclear magnetic resonance spectra of end products from cell suspensions of wild-type L. plantarum NCIMB8826 (A) and LDH-defective L. plantarum TF103 (B) under the following conditions: aerobiosis and 50 mM glucose (a); aerobiosis and 50 mM glucose plus 10 mM citrate (b); anaerobiosis and 50 mM glucose (c); and anaerobiosis and 50 mM glucose plus 10 mM citrate (d). ACN, acetoin; ACT, acetate; BUT, 2,3-butanediol; CIT, citrate; black triangles, resonance of residual glucose; ETH, ethanol; LAC, lactate; MAN, mannitol; PYR, pyruvate; SUC, succinate. Spectra are presented from 15 to 75 ppm. Carbon atoms of the carbonyl groups were also detected, but under our experimental conditions with a 6-s repetition time, those signals were not quantitative.
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TABLE 1. End producta concentrations from glucose and glucose-citrate cometabolism by cell suspensions of wild-type (NCIMB8826) and LDH-defective (TF103) strains of L. plantarum Concn (mM) of end product: Incubation
Strain
Carbon source
Aerobic
a
Citrate
Lactate
Acetate
8 6
4
72 77
6 12
2 14
1 3
11 3
75 82
6
18
4
2
7
TABLE 2. Enzymatic activities detected in cell extractsa from the TF103 and NCIMB8826 strains of L. plantarum Enzyme
Sp act (U/mg of proteinb) of strain: TF103
Pyruvate carboxylase Malate dehydrogenase Malolactic enzyme Mannitol-1 phosphate dehydrogenase LDH 10.
Reference(s) for assays
NCIMB8826 23
6 3 10 173 3 1023
5 3 1023 NAc
25 3 1023 23 3 1023
NA 29 3 1023
NDd
14
29 5 2, 22 4 10
Cell extracts were prepared according to the method described in reference
One unit of activity corresponds to the conversion of 1 mmol of substrate per min. c NA, not applicable because of the high level of LDH activity and large amount of lactate in the wild-type strain, which interfere with malate dehydrogenase and malolactic enzyme assays, respectively. d ND, not detected. b
Acetoin
Mannitol
8 4
35 30
4 ,1
3
20
6
2,3Butanediol
2
Succinate
Pyruvate
2 3
3
2
The production of CO2 and volatile compounds was not measured during this study.
lic acid) (28), and the authors hypothesized the involvement of hydroxyisocaproate dehydrogenase activities. However, we did not detect any hydroxyisocaproate dehydrogenase activity in TF103 cell extracts using several a-keto acids as substrates. As to L-lactate production, one possible pathway could be oxalacetate reduction to L-malate followed by malolactic conversion to L-lactate. The two enzymes responsible for these reactions are malate dehydrogenase and the malolactic enzyme (Fig. 1). They have been reported for L. plantarum and other lactobacilli (2, 32) and have also been detected in TF103 (Table 2). Oxalacetate itself may stem from glycolytic phosphoenolpyruvate or pyruvate via the corresponding carboxylases. These enzymatic activities have been described for several bacterial species (13), but to our knowledge, they have not been reported for lactic acid bacteria. Yet, TF103 extracts showed pyruvate carboxylase activity (Table 2), which is also present in the wild-type strain, but no phosphoenolpyruvate carboxylase activity. The observation of small amounts of succinate (Table 1), another metabolite derived from oxalacetate via malate (Fig. 1), supports this model. Aerobic and anaerobic glucose-citrate cometabolism in LDH-deficient strain TF103. A typical anaerobic pathway frequently observed in lactic acid bacteria is pyruvate formatelyase, which leads to formate accumulation. However, the lack
a
Ethanol
of this compound in buffered cell suspensions from anaerobically grown cultures suggests the absence of this pathway. It must be mentioned that the preparation of cell suspensions included manipulations in the presence of oxygen. Therefore, pyruvate formate-lyase (if any) is likely to have been inactivated, since the enzyme is reported to be highly sensitive to oxygen (8). Most of the end products detected during aerobic and anaerobic glucose-citrate cometabolism were the same as those derived from glucose alone (Table 1). Acetoin remained the major metabolite. We also identified trace amounts of 2,3butanediol resulting from acetoin reduction. Lactate production was largely increased, and all the additional lactate was in the form of the L isomer. This fits the above-described hypothesis of L-lactate production through malolactic fermentation; under these conditions, oxalacetate would arise from citrate (Fig. 1). On the other hand, levels of ethanol and mannitol production were lower in the presence of citrate, which could reflect NAD1 regeneration via other pathways owing to citrate utilization as the external electron acceptor. Small amounts of pyruvate were also detected. Wild-type Lactococcus lactis and Leuconostoc spp. have been reported to excrete pyruvate under conditions in which C4 compounds are formed, i.e., during citrate fermentation (18, 38), and the presence of these compounds has been correlated with an internal accumulation of high concentrations of pyruvate, which very likely occurs in the LDH-defective strain TF103 of L. plantarum. This research was carried out within the framework of the European Community Research Programme ECLAIR with financial contributions from the Commission (contract AGRE-CT90-0041) and from the General Directorate for Research and Technology of the Walloon Region (contract 1580). T. Ferain holds an I.R.S.I.A. fellowship. We are indebted to E. Van Schaftingen and M. Veiga da Cunha for helpful discussions and comments during the course of this work. REFERENCES 1. Bernard, N., K. Johnsen, T. Ferain, D. Garmyn, P. Hols, and J. J. Holbrook. 1994. NAD1-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization. Eur. J. Biochem. 224:439–446. 2. Caspritz, G., and F. Radler. 1983. Malolactic enzyme of Lactobacillus plantarum. J. Biol. Chem. 258:4907–4910. 3. Cavin, J. F., H. Prevost, J. Lin, P. Schmitt, and C. Divies. 1989. Medium for screening Leuconostoc oenos strains defective in malolactic fermentation. Appl. Environ. Microbiol. 55:751–753. 4. Chakravorty, M. 1964. Metabolism of mannitol and induction of mannitol 1-phosphate dehydrogenase in Lactobacillus plantarum. J. Bacteriol. 87: 1246–1248.
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