Biotechnol. R o ~1993, . 9, 113-121
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REVIEW Metabolic Regulation of End Product Distribution in Lactobacilli: Causes and Consequences Ching-Ping TsengtJ and Thomas J. Montville*,tt§ Graduate Program in Microbiology and Molecular Genetics and Department of Food Science, New Jersey Agricultural Experiment Station, Rutgers, the State University of New Jersey, New Brunswick, New Jersey 08903-0231
This review examines the regulation of end product distribution in Lactobacillus plantarum and other lactobacilli, the factors that influence this distribution, and the bioenergetic consequences of end product distribution. Similarities to and differences from other genera in the lactic acid bacteria are described. Lactobacilli use many different transport systems to obtain nutrients. Carbohydrates and organic acids are transported in cells by specific phosphoenolpyruvate phosphotransferases (PEP-PTS) or permeases. The carbohydrates are then metabolized to different end products through a common key intermediate, pyruvate. The ability of lactobacilli to produce various end products depends on species, strains, genetic capacity, expression of enzyme activity, sugar structure, and environmental conditions. The regeneration of the NAD+ required for continued glycolysis is a key regulatory factor of end product distribution. The excretion of protons with acidic end products can directly generate a proton motive force and it also contributes to intracellular pH homeostasis. Anionic precursor-product exchange systems can also generate a proton motive force if the exchange is not electroneutral.
Contents I. Introduction 113 11. General Characteristics of Lactobacilli 113 111. Transport and Metabolism 114 A. Sugars 114 B. Organic Acids 115 IV. Physiological Regulation of 116 Carbohydrate Metabolism A. Regulation of Lactate Dehydrogenase 116 B. Regulation by Environmental pH 117 C. Regulation by Oxygen 117 V. Bioenergetics of Lactobacilli 117 VI. Conclusions 118 I. Introduction Conversion of carbohydrates to lactate may be the most important fermentation in food technology. Lactate’smild acidic taste does not mask weaker aromatic flavors. The addition of lactic acid to foods acidifies them, enhances other flavors, and inhibits microbial growth. Lactobacilli produce other catabolites such as diacetyl, acetoin, 2,3butanediol, formate, and acetaldehyde. These also contribute to the flavor of dairy products (Marshall, 1987).
* Corresponding author, (908) 932-9663.
Graduate Program in Microbiology and Molecular Genetics. address: Department of Microbiology and Molecular Genetics, 5304 Life Science Building, 405 Hilgard Ave., University of California, Los Angeles, CA 90024-1489. Department of Food Science. +
1 Current
8756-7938/93/3009-0113$04.00/0
Lactobacilli are used in the fermentation of dairy (Gilliland, 1985),vegetable (Fleming et al., 19851,and meat (Bacus and Brown, 1985)products. Their hardiness, acid tolerance, wide range of substrates, and aerotolerance are significant advantages in the manufacture of foods having market values in the tens of billions of dollars (Chassy, 1985). Genetic manipulation of lactobacilli can develop strains that are faster growing, are more efficient in their biotransformations, and have more defined metabolic outcomes. However, because catabolic products are made by multienzyme pathways coded by a multitude of genes, genetic manipulationof end products is complicated unless appropriate rate-limiting enzymes can be identified. Thus, there is a continued need to study the metabolicregulation of catabolism in lactobacilli. In addition, knowledge of how cells cope with unfavorable (i.e., acidic, alkaline, oxidized, nutrient-depleted) environments would allow specific fermentations to be channeled toward more desirable end products. There are several excellent reviews of lactic acid bacterial metabolism and physiology (Kandler, 1983;Kashket, 1987;Thompson, 19871,but most of them focus on lactococci or streptococci. This review describes how lactobacilli transport, metabolize, and ration their substrates among alternate pathways under different environmental conditions.
11. General Characteristics of Lactobacilli Lactobacilli are Gram-positive, rod-shaped lactic acid bacteria which are associated with humans, animals, dairy products, fermented beverages, and plant material. Lactobacilli are usually classified as being either homo- or heterofermentative. Homofermentative lactobacilli pro-
@ 1993 American Chemical Society and American Institute of Chemical Engineers
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Dr.Ching-Ping Tseng, a native of Taipei, Taiwan, received his B.S. degree in chemistry from Soochow University and his M.S. degree in biochemistry from the National YangMing Medical College, both in Taipei. He served as an instructor a t Shih-Chien College prior to entering the Rutgers University/Robert Wood Johnson University of Medicine and Dentistry Joint Program in Microbiology and Molecular Genetics. Here he served as a teaching assistant and finally a graduate assistant in Prof. Montville’s laboratory where he conducted his doctoral research. His dissertation on enzymatic regulation of glucose catabolism by Lactobacillus plantarum and its bioenergetic consequences resulted in four publications and provided the impetus for writing this review. An accomplished pastel artist, Dr. Tseng is currently a postdoctoral research associate in the laboratory of Prof. Robert Gunsalus a t the Department of Microbiology and Molecular Genetics a t the University of California-Los Angeles.
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duce only lactate, whereas heterofermentativelactobacilli produce a t least 50% lactate from the fermentation of glucose. Homofermentative bacteria metabolize one molecule of glucose via the Embden-Meyerhoff-Parnas (EMP) pathway to two molecules of pyruvate and concurrently generate two molecules of ATP and NADH (Kandler, 1983;Kandler and Weiss, 1986). The pyruvate is reduced to lactate in a reaction linked with NADH oxidation. This regenerates the NAD+ required for continued glycolysis. Heterofermentative bacteria metabolize one molecule of glucose via the 6-phosphogluconate pathways to produce one molecule of carbon dioxide, one molecule of ethanol (or acetate), and one molecule of lactate. The presence or absence of fructose1,6-bisphosphate (FBP) aldolase is responsible for the difference between homo- and heterofermentative organisms. Homofermentative lactobacilli have an FBP aldolase. Heterofermentative organisms lack it, but do have a phosphoketolase to split hexoses and pentoses (Kandler, 1983; Gottschalk, 1986). Normally homofermentative lactobacilli that ferment pentoses to lactate and acetate through an inducible phosphoketolase are considered facultatively heterofermentative (Schleifer, 1987). Lactobacillus plantarum is a member of this group. Although most lactobacilli are aerotolerant or microaerophilic, anaerobicconditions usually provideoptimal growth. Lactobacilli have no cytochromes and are superoxide dismutase and catalase negative. However, most have flavine-containing oxidases and peroxidases which oxidize NADH using oxygen as the final electron acceptor (Kandler and Weiss, 1986). Thus, L. plantarum produces more biomass at low oxygen transfer rates than under strictly anaerobicconditions (Montvilleand McFall, 1989). Some lactobacilli require manganese as a cofactor for enzyme systems such as lactate dehydrogenase, RNA polymerase, and manganicatalase (Archibald, 1986). Aerotolerance and oxygen utilization in lactobacilli correlate with high intracellular manganeselevels (Gotzet al., 1980b; Archibald and Duong, 1984). L. plantarum uses concentrations of intracellular manganese up to millimolar levels to scavenge superoxide radicals and degrade hydrogen peroxide (Archibald and Fridovich, 1981a,b). 111. Transport and Metabolism
Dr. Thomas J. Montville is Professor of Food and Fermentation Microbiology and Director of the Graduate Program in the Department of Food Science a t Rutgers, the State University of New Jersey. His laboratory studies the physiology of lactic acid bacteria and their use in food systems, both to impart desirable characteristics and to inhibit foodborne pathogens. Prof. Montville is a frequent consultant and lecturer in the food industry, has published more than 100papers and abstracts in these areas, serves on the editorial board of the Journal of Food Protection, and is coeditor of the Journal of Food Safety. Dr. Montville received his Ph.D. degree from the Massachusetts Institute of Technology and his B.S. degree from Rutgers, and he has recently been elected a fellow of the American Academy of Microbiology. He enjoys playingracketball with students and faculty, lives near campus with his wife Nancy and their children Christopher, Rebecca, and Matthew, and is a member of the American Society for Microbiology,the Institute of Food Technologists, the Society for Industrial Microbiology, and the American Association for the Advancement of Science.
A. Sugars. Active transport systems in lactic acid bacteria are classified according to their mode of energy coupling (Konings, 1983; Poolman et al., 1987a,b) (Figure 1). In ATP-dependent transport systems, transport is driven by the energy released from the hydrolysis of highenergy phosphate bonds. In secondary transport systems, transport is coupled to proton motive force. In group translocation systems, transport of sugars is catalyzed by a group of enzymes using phosphoenolpyruvate as the energy source; the solute is phosphorylated during translocation. Fermentable sugars are usually transported by group translocation (Chassy and Thompson, 1983a,b; Thompson, 1987), whereas most amino acids are transported by secondary transport systems (Driessen, 1989; Shay et al., 1988). Lactic acid bacteria transport lactose using a phosphoenolpyruvate-lactose phosphotransferase system (PEPPTS) or a lactose permease with subsequent intracellular phosphorylation. The lactose phosphate is cleaved by phospho-@-galactosidaseto yield glucose and galactose 6-phosphate. The resulting glucose is metabolized via glycolysis, and the galactose 6-phosphate is metabolized through the D-tagatose 6-phosphate pathway to form dihydroxyacetone phosphate and glyceraldehyde3-phos-
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Bbtechnol. Reg, 1993, Vol. 9, No. 2 Citrate
h
Citrate /yase
M&M~Z+
Acetate
,--
TRANSPORT
II
Ant'pJn
A'
342 lSECONDARYl TRANSPORT
PEP
\
Pyruvate
9
Oxaloacetate
J1oxa/oacett;
hcarboxy/ase
Acetaldehyde-TPP
L k +
l o - a c e t d a c t s t e synthase H+
a-acetolactate
a-acetdactate decarboxylase
Sugar GRWP TRANSLOCATION
Figure 1. Schematic presentation of various transport systems in lactic acid bacteria (adapted from Konings, 1983). phate. When lactose is transported by a specific permease and hydrolyzed by/??-galactosidase (Premi et al., 1972;Toba et al., 19811, the resulting galactose is phosphorylated, converted to glucose 6-phosphate, and fermented via glycolysis to pyruvate. /??-Galactosidaseis inducible in these cells by galactose and lactose (Killara and Shahani, 1976). Glucose acta as it does in Escherichia coli;catabolite repression prevents galactose or lactose from entering the cell (Hasan and Durr, 1974). Lactobacilli transport most sugars and oligosaccharides by specific permeases and phosphorylate them intracellularly. Oligosaccharides are hydrolyzed by glycosidases prior to the phosphorylation of the resulting monosaccharides. However, Lactobacillus casei transports lactose and galactose by a PEP-PTS which is induced by galactose and lactose but repressed by galactose 6-phosphate and high glucose concentrations (McKay et al., 1970; Chassy and Thompson, 1983a,b). Transport of glucose in lactobacilli is either by an active transport permease system or the PEP-PTS system (Romano et al., 1979). L. casei has PEP-glucose phosphotransferase and glucokinase activities, whereas Lactobacillus brevis and Lactobacillus buchneri, which utilize the pentose phosphoketolase pathway, have only glucokinase activity. Lactobacillus helveticus metabolizes both glucose and galactose moieties of lactose, but Lactobacillus bulgaricus, Lactobacillus lactis, and Lactobacillus acidophilus only metabolize the glucose and excrete the galactose (Hickey et al., 1986).All four species contain /??-galactosidaseactivity, but no phospho-&galactosidase activity. L. bulgaricus and L. helveticus have PEP-glucose PTS systems, but there is no evidence for PEP-lactose or PEP-galactose PTS systems. In L. plantarum, glucose is transported by a PEPglucose phosphotransferase and oxidized via glycolysis to two molecules of pyruvate (Romano et al., 1979). A net yield of two molecules of ATP is formed by substratelevel phosphorylation while two molecules of NAD+ are reduced. Since pyruvate is the key intermediate in lactobacilli catabolism, the diversity of end products produced depends on reactions essential for continued glycolysis in L. plantarum. The primary mechanism for NAD+ regeneration is by the nicotinamide adenine dinucleotide linked lactate dehydrogenase (LDH) mediated reduction of pyruvate to lactate (Garvie, 1980). When LDH is suppressed, homofermentative organisms grow slowly. Therefore, LDH is considered to be the most important enzyme in energy production and cell growth.
Diacetyl
1
hacety/ reductase
I
co2
Acetoin
Figure 2. Metabolism of citrate and synthesis of diacetyl and acetoin in lactic acid bacteria (adapted from Speckman and Collins, 1973). When alternate hydrogen acceptors are present, pyruvate is available to form acetate, ethanol, acetoin, and diacetyl (Collins, 1972). If exogenous pyruvate is added to L. plantarum pH-controlled cultures, it enters the catabolic pool without a requirement for NAD+ regeneration and is quantitatively converted to acetoin (Montville et al., 1987). B. Organic Acids. The citrate transport system and its relationship to diacetyl and acetoin production have been extensively studied (Figure 2). Citrate is transported by citrate permease; its uptake increases as the external pH decreases (Cogan et al., 1981). Glucose and lactose stimulate citrate utilization, but totally inhibit acetoin production in glucose- and lactose-grown cells (Cogan et al., 1981; Cogan, 1987). Intracellular citrate is degraded by citrate lyase to acetate and oxaloacetate. Oxaloacetate is then decarboxylated to form pyruvate (Kempler and McKay, 1979). Although no ATP is produced, this pathway allows the organism to produce pyruvate without incurring an NAD+ debt. The *excess" pyruvate may be used in the synthesis of diacetyl and acetoin. Most strains of lactobacilli cannot metabolize citrate as their sole carbon source (Radler and Brohl, 1984),but cometabolize citrate in the presence of another carbon source (Drinan et al., 1976;Kenness et al., 1991). In commercial fermentations, citrate is frequently added to milk to enhance diacetyl synthesis. Pyruvate is the key intermediate in lactobacillic catabolism. When pyruvate is dissimilated through enzymes other than LDH, an alternate mechanism of NAD+ regeneration must be activated and end products other than lactate are produced (Collins, 1972; Drinan et al., 1976). In addition, because an exogenous source of pyruvate does not carry a glycolytically incurred NAD+ debt, the free pyruvate can be converted to stoichiometrically significant quantities of acetoin (El-Gendy et al., 1983a,b; Hickey et al., 1983; Montville et al., 1987). L. plantarum cells grown in medium containing exogenous pyruvate divert pyruvate away from lactate to acetoin. On a molar basis, pyruvat,e utilization increases twice as fast as acetoin production, reflecting the 2:l stoichiometry of pyruvate incorporation into acetoin (McFall and Montville, 1989). When pyruvate is converted to acetoin, it is decarboxylated and combined with hydroxyethylthiamine pyrophosphate (TPP) to form an active acetaldehyde-TPP (Figure 2). The acetaldehyde-TPP complex then con-
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piDH
Glucose
NADNADH
Lactate
Lactate synthase
Acetolacate
/-AcetypA
Formate
Slosynthess
Acetate
4 NADH + H ?+
NAD+
NAD'
H202 Ethanol
Figure 3. Composite catabolic pathways for pyruvate dissimilation in lactic acid bacteria.
denses with another molecule of pyruvate to form a-acetolactate. a-Acetolactate is consecutivelydecarboxylated by a-acetolactate decarboxylaseto form acetoin or oxidized todiacetyl. The diacetyl can be further reduced to acetoin and 2,3-butanediol (Hickey et al., 1983) (Figures 2 and 3). The maximum formation of acetoin from a-acetolactate by L. casei is between pH 5 and 6 (Branen and Keenan, 1971),the optimal pH for a-acetolactate synthase activity (Cogan, 1984). Pyruvate is actively transported in L. casei subsp. rhamnosus by a constitutive pyruvate-specific carrier (Benitode Cardenas et al., 1987). L. plantarum transports pyruvate by a symport carrier system, which is also constitutive and specific (Tsau, 1991). When pyruvate and a proton(s) are cotransported, the intracellular pH drops (Tsau et al., 1992). The decrease of intracellular pH increases a-acetolactate synthase and acetoin dehydrogenase activities (Tseng and Montville, 1992a). This causes the excess intracellular pyruvate, which results from the accumulation of transported pyruvate, to be converted to mostly acetoin and some lactate with a concurrent increase in intracellular pH (Tsau et al., 1992). This conversion of an acidic end product to a neutral one functions as a mechanism to maintain intracellular pH homeostasis. Pyruvate can also be converted to formate and acetate via the pyruvate formate lyase (PFL, Figure 3) pathway in Lactococcus lactis, Lactobacillus cremoris, and L. bulgaricus (Rhee and Pack, 1980; Thomas et al., 1980).If the acetyl-coA is further reduced to acetaldehyde and then to ethanol, NAD+is regenerated (Condon, 1987).PFL is extremely oxygen-sensitive and is inhibited by glyceraldehyde 3-phosphate as well as by dihydroxyacetone phosphate. Other organic acids, such as malate, fumarate, and tartrate, are degraded via oxaloacetate and pyruvate to CO:! and lactate or acetate (Radler and Brohl, 1984). Although the malolactate fermentation is usually associated with the deacidification of red wines by Leuconostoc oenos, it can also be affected by pediococci, lactobacilli, streptococci, and lactococci (Kunkee, 1976). Malate can be fermented directly to L-lactate and COz by the malolactic enzyme found only in lactic acid bacteria. The malolactic reaction increases a culture's growth rate even though no ATP or NADH is generated (Renault et al., 1988). A t low pH values, malate is used preferentially
over sugars (Champagne et al., 1989) and appears to compete with citrate for transport (McCord and Ryu, 1985). The malolactic enzyme is relatively nonspecific (Davis et al., 1986) and converts a dicarboxylic acid (malate) to a monocarboxylic acid (lactate). As a result, both medium and intracellular pH increase. In his excellent review of microbes and membrane biology, Maloney (1990)suggests that the malate2--lactate1-couple generates energy via a product-precursor exchange model. This model is discussed in more detail below. Even in the absence of a specific malate-lactate exchanger, the use of the malate gradient to drive transport may allow cells to derive energy from the malolactic fermentation (Olsen et al., 1991). Energy could also be generated by coupling the translocation of protons through the membrane with the end product efflux (Renaukt et al., 1988). The transport system of L-malate in L. plantarum is inducible and can be driven by an artificial proton gradient. The transport and excretion of lactic acid in lactobacilli occurs via a specific carrier-mediated facilitated diffusion system (Gatje et al., 1991). Most lactic acid bacteriaexcrete lactate as the main end product of carbohydrate metabolism and do not subsequently use the lactate as a growth substrate. However, aerated L. plantarum can convert lactate to acetate (Murphy and Condon, 1984a; Murphy et al., 1985). Enterococcus faecium, Lactobacillus delbruekii, and L. casei growing under aerobic conditions also use lactate (Hager et al., 1954; Strittmatter 1959; London, 1968). L. plantarum metabolizes lactate even under anaerobic conditions after prolonged incubation when glucose is absent (Montville et al., 1987; Lindgren et al., 1990).
IV. Physiological Regulation of Carbohydrate Catabolism A. Regulation of Lactate Dehydrogenase. Lactobacilli produce both L- and D-lactate. Some homofermentative lactobacilli (e.g., L. curvatus; Garvie, 1980) produce more L-lactate in the early growth phase. After L-lactate is accumulated, an inducible racemase converts L-lactate into D-lactate until equilibrium is reached. L. plantarum grown without pH control contains both L-LDH and D-LDHthat differ in stereospecificity; the proportion of D-lactate and L-lactate production does not change (Dennis and Kaplan, 1960; Mizushimaet al., 1964;Gasser, 1970; Kandler, 1983). NADH-dependent lactate dehydrogenase (iLDH) which catalyzes the reaction of lactate back to pyruvate has been isolated from lactobacilli, but theK, for lactate is relatively high (Snoswell,1963;Doelle, 1971) and the reaction is confined to the late stationary phase of growth (Montville et al., 1987). The influence of LDH activity on end product distribution can be seen in other systems. Strains defective in either LDH or the lactose phosphotransferase system and/or phospho-8galactosidase express a heterofermentative pattern (Deme ko et al., 1972;McKay and Baldwin, 1974). LDH mutants of L. casei also channel pyruvate to increase diacetyl formation (Miyamoto et al., 1983). Homofermentative organisms which produce heterofermentative catabolites in glucose-limitedchemostats are regulated by LDH activity. For example, L. casei growing at high dilution rates has high intracellular fructose 1,6bisphosphate levels. Since fructose 1,6-bisphosphate activates LDH, only lactate is produced. Cells growing at low dilution rates have less intracellular fructose 1,6bisphosphate available to activate LDH and more pyruvate is diverted to form other end products (De Vries et al., 1970). Fructose 1,6-bisphosphate plays a similar regula-
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tory role in clostridia (Hadggstrom, 1986)and streptococci (Yamada and Carlsson, 1975). Other chemostat studies also revealed shifts from homo- to heterofermentative metabolism in L. lactis and L. cremoris (Thomas et al., 1979,1980;Thomas and Turner, 1981;Fordyce et al., 1984). However,reducing the dilution rate does not divert lactate to other products in L. plantarum ATCC 8014 (McFall, 1988) or L. lactis ML8 (Thomas et al., 1979). B. Regulation by Environmental pH. Lactobacilli grow over a wide pH range in the presence of organic acids. Changesfrom the homofermentative to heterofermentative pattern can be induced by alkaline conditions (Campbell and Gunsalus, 1944;Rhee and Pack, 1980). L. bulgaricus synthesizes less LDH under alkaline conditions, suggesting that the LDH concentration controls pyruvate metabolism (Rhee and Park, 1980). The high LDH synthesis in acidic culture conditions, together with the alkaline optima of enzymes related to the phosphoroclastic split such as pyruvate formate lyase (Lindmark et al., 19691, acetate kinase (Satchel1 and White, 1970), acetaldehyde dehydrogenase (Rudolph et al., 19681, and phosphotransacetylase (Hibbert et al., 1971),may explain why L. bulgaricus NLS-4 shifts from homofermentative to heterofermentative metabolism when the environmental pH changes from acidic to alkaline. In contrast to L. bulgaricus,L. plantarum has increasing LDH activity and an increasing specific rate of lactate formation at higher pH values (Tsengand Montville, 1990). Acetoin production decreases with increasing pH as it does in the other lactic acid bacteria because of the lower acetoin dehydrogenase and a-acetolactate synthase activities (Cogan et al., 1984). The high pH optimum of acetate kinase causes L. plantarum growing under alkaline conditions to make more acetate at the expense of neutral compounds (McFall and Montville, 1989; Tseng and Montville, 1990). The ability to shift between the production of neutral and acidic compounds may help L. plantarum make more ATP and maintain pH homeostasis (Tseng et al., 1991). C. Regulation by Oxygen. Most lactobacilli are strictly fermentative, but aerotolerant (Kandler and Weiss, 1986). A number of inducible flavoprotein oxidases are responsible for the cell’s interaction with oxygen. These enzymes mediate the use of oxygen as an electron acceptor and spare the use of metabolic intermediates such as pyruvate or acetaldehyde as electron acceptors. Consequently, energy, biomass, end products, and substrate specificity are affected (Condon, 1987). Under aerobic conditions, pyruvate can be dissimilated to acetate through an oxygen-inducible pyruvate oxidase (Lloyd et al., 1978;Hickey et al., 1983;Murphy and Condon, 1984a;Sedewitz et al., 1984a,b;Tseng and Montville, 1990). In the first step of this pathway, which might be shared with acetoin synthesis, pyruvate is converted to the acetaldehyde-TPP complex (Figure 3). However, pyruvate can be directly phosphorylated to acetyl phosphate. Acetate kinase converts acetyl phosphate to acetate, yielding one ATP by substrate-level phosphorylation. Because two additional ATP molecules are formed per molecule of hexose fermented to acetate, higher specific growth rates per molecule of hexose and greater cell yields are expected. However, growth of some strains is stimulated in aerobic cultures (Driar and Collins, 1973;Murphy and Condon, 1984b),rrhereas others are inhibited (Archibald and Fridovich, 1981a). All lactic acid bacteria contain NADH oxidase. Lactobacilli have two cytoplasmic non-hemc NADH oxidases. One catalyzes the reduction of oxygen to water and the
other catalyzes the reduction of oxygen to Hz02. Because NADH oxidase and the dismutation of superoxide radical produce H202, aerated cultures require an NADH peroxidase or catalase to convert HzOz to HzO (Stoll and Blanchard, 1988). Under aerobic conditions, NADH oxidase and NADH peroxidase compete with LDH for NAD+regeneration. NADH peroxidase not only provides redox balance but also detoxifies the intracellular H202 (Gotz et al., 1980a). L. plantarum can use oxygen as an electron acceptor even though it lacks a complete heme-linked electron transport system (Strittmatter, 1959; London, 1976). Under aerobic growth conditions, L. plantarum consumes oxygen in the presence of glucose, lactate, or pyruvate as substrates (GBtz, et al., 1980a,b; Murphy and Condon, 1984b; Murphy et al., 1985; Montville and McFall, 1989; Tseng and Montville, 1990). The specific activities of pyruvate oxidase, acetate kinase, NADH oxidase, and NADH peroxidase are increased by oxygen. This results in the production of acetate in addition to lactate (Murphy and Condon, 1984a;Sedewitz et al., 1984b; Murphy et al., 1985; Tseng and Montville, 1990). In complex medium with a low glucose concentration (2.78 mM), L. plantarum has similar growth rates under aerobic or anaerobic conditions, but has higher cell yields during aerobic growth (Yousten et al., 1975). In contrast, at a high glucose concentration (50mM), L. plantarum decreases its growth rate during aerobic incubation (Archibald and Fridovich, 1981a); on prolonged incubation the cell yield in aerobic cultures is at least equal to that in anaerobic cultures (Murphy and Condon, 1984b). Decreased growth rates in the presence of oxygen are associated with superoxide radical or the accumulation of Hz02. The H202 is produced by high specific activities of NADH oxidase or pyruvate oxidase (Murphy et al., 1985). In addition, the acetate kinase activity of L. plantarum increases under aerobic conditions; this also increases acetate production (Tseng and Montville, 1990;Tsengand Montville, 1992b). Aerobic metabolism of sugars to acetate rather than lactate increases the amount of ATP formed through substratelevel phsophorylation. Lactic acid bacteria which have an oxygen-insensitive mechanism of acetate synthesis benefit from the extra ATP generated by producing more biomass and possibly by growing faster.
V. Bioenergetics of Lactobacilli The energy-producing capabilities of bacteria are frequently described only in terms of the ATP produced. However, ATP constitutes only one portion of a cellular economy which has as interconvertible currencies both ATP and proton motive force. Most bacteria establish and maintain an electrochemical gradient of protons across their cytoplasmic membranes, such that the interior of the cell is alkaline and the exterior acidic. The subsequent downhill flux of protons (with the proton gradient from the outside to the inside of the cell) can be linked to the uphill transport of another compound against a concentration gradient. This is how secondary transport systems link the proton gradient to the performance of useful work (Figure 1). The proton gradient is interconvertible with ATP by the H+-translocating ATPase (Kashket, 1987). Aerobes generate the proton gradient using the electron transport system, but strictly fermentative lactobacilli lack a complete electron transfer system that can function as a proton pump. In these bacteria, the ATPase functions in the direction of hydrolysis and H+ extrusion (Figure 4). This generation of proton motive force by ATP hydrolysis consumes the limited ATP formed by substrate-level phosphorylation, making less ATP available for biosyn-
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118 acid
+
7
alkaline
ATPase PROTON
EXCHANGE lactate.'. malate''-
I
lactate-
nH+
acetate'
nH+
I
!=#+Id F N O PRODUCT EFFLUX]
Figure 4. Mechanisms by which lactobacilli generate proton motive force when lactate and acetate are produced by the fermentation of glucose.
thesis (Hellingwerf and Konings, 1985). It now appears that these organismscan use two ATP-sparing mechanisms to generate proton gradients. These mechanisms are described by the end product efflux and anion exchange models presented below. Many studies suggest that lactic acid bacteria have developed systems for the efflux of protons in symport with catabolic end products (Gatje et al., 1991;Michels et al., 1979; Ten Brink and Konings, 1982; Ten Brink et al., 1985; Simpson et al., 1983; Tseng et al., 1991). In active transport, symports use the flow of protons down a proton concentration gradient to drive the transport of a substrate against a substrate concentration gradient. This results in the active transport of the substrate at the expense of the proton gradient. During end product efflux,the reverse occurs. The flow of a catabolic product down its concentration gradient is coupled to the flow of protons against the proton gradient. This results in the generation of a proton gradient at the expense of the catabolite gradient. Membrane potential is generated by this mechanism only when a net positive charge is translocated (Le., two H+ accompany one monovalent anion across the membrane; Figure 4). Thus, the energy of the end product gradient is converted into an electrochemical proton gradient. Since catabolic end products are continuously produced during fermentation, a continuous efflux of end products occurs and continuous generation of proton motive force can be achieved. As a result, carrier-mediated efflux of end products can make a significant contribution to the metabolic energy derived from fermentation. The contribution of end product efflux to proton motive force generation has been studied in homofermentative lactic acid bacteria such as L. cremoris and in E . coli vesicles (Ten Brink and Konings, 1980). Depending on the H+: lactate stoichiometry and the extracellular lactate concentration, as much as one additional ATP can be generated by this carrier-mediated efflux process. This constitutes an energy gain of 50 3' 6 (Ten Brink et al., 1985). The energy recycling model may also apply to other organic acids (Ten Brink and Konings, 1986). L. plantarum cells produce acetate in addition to lactate under alkaline and aerobic conditions. The bioenergetic consequence of this catabolic shift is that acetate coexcretes more protons than lactate does. Both H+:lactate and H+:acetate stoichiometry values increase in alkaline and aerobic conditions. L. plantarum pumps out more protons with end products to maintain intracellular pH homeostasis and generates proton gradients under aerobic and alkaline conditions. During the transition from acid to alkaline conditions, the
stoichiometry of protons:acetate approaches 3, which would spare one ATP (Tseng et al., 1991). An anion exchange system could also indirectly establish a proton gradient. Maloney (1990) postulated a general model whereby the antiport of a divalent precursor anion with a monovalent product anion would generate a membrane potential. He suggested that the malateZ-lactatel- couple might generate energy in this fashion. The consumption of an intracellular proton during the decarboxylation of malate to lactate has the net effect of increasing the proton gradient (interior alkaline) by one proton per molecule of malate metabolized. Experimentally, anion exchange systems can be misidentified as proton symport systems. There is some controversy over which systems function under a given set of conditions in a given bacterial species.
VI. Conclusions Although the ascendancy of molecular genetics has caused research on metabolic regulation to become unfashionable (and largely unfundable), considerable advances have been made in the last 30 years. We have attempted to demonstrate that the catabolism of lactobacilli is much more subtly regulated than the traditional classification "homofermentative" suggests. L.plantarum has mechanisms to convert pyruvate to products other than lactate. These catabolic shifts help maintain pH homeostasis, take advantage of transient aeration, and spare significant quantities of ATP through the extrusion of protons linked to end product excretion. Anion exchange models and new insight on the malolactic fermentation suggest that these anaerobes are not as bereft of energy as previously thought.
Acknowledgment This is manuscript D-10112-1-92of the New Jersey State Agricultural Experiment Station, supported by the State of New Jersey and U.S.Hatch funds. We gratefully acknowledge the contributions of M. B. Meyer, A. Hsu, S. McFall, and J. L. Tsau to our understanding of this subject. We thank A. Crandall for redrawing the figures and also thank G. Carman for many stimulating discussions and challenging questions during the course of our research.
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Additional Reading Booth, I. R. Regulation of Cytoplasmic pH in Bacteria. Microbiol. Rev. 1985,49,359-378. Harold, F. M. The Vital Force, A Study of Bioenergetics; W.H. Freeman and Co.: New York, 1986. Maloney, P. C.; Ambudkar, S. V.; Anantharam, V.; Sonna, L. A.; Varadhachary, A. Anion-Exchange Mechanisms in Bacteria. Microbiol. Rev. 1990, 54, 1-17. Accepted July 3, 1992.
