Ph Homeostasis in Lactic Acid Bacteria

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Faculty Publications in Food Science and Technology Food Science and Technology Department

September 1993 pH Homeostasis in Lactic Acid Bacteria

Robert W. Hutkins University of Nebraska-Lincoln, [email protected]

Nancy L. Nannen University of Nebraska-Lincoln

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Hutkins, Robert W. and Nannen, Nancy L., "pH Homeostasis in Lactic Acid Bacteria" (1993). Faculty Publications in Food Science and Technology. 28. https://digitalcommons.unl.edu/foodsciefacpub/28

This Article is brought to you for free and open access by the Food Science and Technology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications in Food Science and Technology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. pH Homeostasis in Lactlc Acld Bacterial

ROBERT W. HUTKINS and NANCY L. NANNEN Department of Food Science and Technology University of Nebraska-Lincoln Lincoln 68583.0919

ABSTRACT especially important concern for lactic acid The ability of lactic acid bacteria to bacteria used as dairy starter cultures because regulate their cytoplasmic or intracellular these obligate acid-producers must cope with pH is one of the most important physio- low pH, high acid environments during ordi- logical requirements of the cells. Cells nary growth in milk. This paper reviews the unable to maintain a near neutral in- effects of low pH, and in particular, low intracellular pH during growth or storage tracellular pH (pHin), on growth and metabo- at low extracellular pH may lose viabil- lism of lactic acid bacteria and discusses how ity and cellular activity. Despite the im- cytoplasmic pH regulates cell metabolism and portance of pH homeostasis in the lactic how cytoplasmic pH is regulated in the lactic acid bacteria, however, an understanding acid bacteria. of cytoplasmic pH regulation has only recently begun to emerge. This review GROWTH OF LACTIC ACID describes the specific effects of low pH BACTERIA IN MILK on lactic acid bacteria, reports recent re- In general, growth of lactic acid bacteria search on the physiological role of in- continues as long as 1) carbohydrates, amino tracellular pH as a regulator of various acids, and other nutrients are available; 2) metabolic activities in lactic acid bacte- toxic or inhibiting compounds, such as hydro- ria, and presents the means by which gen peroxide, are removed, degraded, or lactic acid bacteria defend against low diluted; or 3) the hydrogen ion concentration is intracellular pH. Particular attention is maintained above the level that a specific devoted to the proton-translocating strain can tolerate. For example, growth of ATPase, an enzyme that is largely Lactococcus lactis ssp. lactis, Lactococcus lac- responsible for pH homeostasis in fer- tis ssp. cremoris, and Streptococcus ther- mentative lactic acid bacteria. mophilus in milk occurs until the pH reaches (Key words: pH, pH homeostasis, lactic approximately 4.5, despite nonlimiting concen- acid bacteria) trations of nutrients and the relative absence of inhibitory compounds (21, 61. 98). Low pH Abbreviation key: pHout = extracellular pH, (or high lactic acid) is frequently growth- pHi, = intracellular pH, ApH = pH gradient, limiting for lactic acid bacteria that are grown H+-ATPase = proton-translocating ATPase. in milk or in weakly buffered bacteriological media (12, 61). INTRODUCTION Although lactic acid bacteria may frequently be isolated from acid habitats (69), The means by which microorganisms toler- such sour and are commonly thought ate variations of environmental pH been an as milk, has to favor low pH environments, except for cer- active area of investigation [for reviews, see tain Lactobacillus species, lactic acid bacteria (16, 41, 46, 51, 76)]. pH homeostasis is an are probably best characterized as neutrophiles. For example, optimal growth rates for the mesophilic dairy starter cultures, L. lactis ssp. Received February 10, 1992. kactis and L. lactis ssp. cremoris have been Accepted April 28, 1992. reported to occur within an external or ex- Published as Paper Number 9795, Journal Series Nebraska Agricultural Experiment Station, Lincoln tracellular pH (pHout) range of 6.3 to 6.9 (12, 685834919, 13,37). The optimal pHout for the thermophilic

1993 J Dairy Sci 76:2354-2365 2354 SYMPOSIUM: DAIRY STARTER CULTURES 2355 lactic acid bacterium S. themphilus was be- ied the damage to L.. fuctis ssp. fuctis ML3 tween 6.5 and 7.5 (9, 72). Among the lactic resulting from growth at low pH. Maximum acid bacteria used as dairy starters, only the specific growth rates occurred at pHout 6.3. lactobacilli (Lactobacillus helveticus and Lac- Cell damage, determined as the ratio of the tobacillus delbrueckii ssp. bulgaricus) appear transient growth rate to the normal growth rate, to grow optimally at acid pH; maximal growth began to occur at medium pH or pbut5.0, and occurs at pH 5.5 to 5.8 (9, 95). the specific growth rate approached zero at Not only do most lactic acid bacteria grow pHout 4.0. A reduction in the specific activity more slowly at low pH, but acid damage and of the enzymes hexokinase and acetate kinase, loss of cell viability may also occur in cells as well as the overall glycolytic activity, also held at low pH. In fermented dairy products, occurred at pH 4.0. Although pHin was not such as yogurt or cultured buttermilk, whether measured, Harvey (37) suggested that the cells the lactic acid bacteria are viable or injured by were able to maintain constant pHh as long as the lactic acid and low pH environment, once pbutwas >5.0. Many other workers (18, 55, the desired pH is reached frequently is techno- 72, 98) have reported that growth of lactic logically irrelevant. Moreover, inhibition of the streptococci and lactococci decreases sharply starter culture by lactic acid and low pH acts to when the medium pH reaches 5.0. Similarly, prevent, in part, overacidification of the fin- pH-induced damage also occurred in the fecal ished product. In the production of yogurt, for coccus, Enterococcus fuecaZis (formerly Strep- example, fermentation lowers the pH of the tococcus faeculis). Below pH 5.0, derangement milk from 6.5 to between 4.0 and 4.5, and the of membrane structures and solute leakage oc- milk coagulates. Unrestricted growth and fer- curred (60). Although the changes were revers- mentation in yogurt (or postacidification) by ible in both L Zuctis ssp. lactis and E. faeculis, the lactic culture may result in excessive acid a lag in fresh media occurred following ex- and flavor defects. In other cases, however, posure to these low pH (37, 60). maintenance of culture viability under acid Bender et al. (1 1) studied the effect of gross conditions is desired and necessary. Cultures membrane damage caused by acidification in and culture adjuncts (e.g., Lactobacillus various oral streptococci. Damage was as- acidophilus) that are added to yogurt for ther- sessed by measurement of the release of mag- apeutic value must remain viable during stor- nesium ions from cells that were incubated at age at low pH and, ultimately, must survive specific pH at room temperature. Magnesium the acid stomach environment. Mixed genus, was not lost from Streptococcus mutuns, Strep- thermophilic cultures used for Italian cheese tococcus sanguis, and Streptococcus salivarius manufacture (i.e., S. themphilus and Lb. hel- at pH near neutrality, but magnesium efflux veticus) are usually propagated or cultivated occurred after the pH was lowered to <4.0. At together; that the former is more acid-sensitive pH 2 or 3, release of magnesium was rapid and than the latter may result in variable strain extensive. In contrast, Lactobacillus casei ratios. In the industrial production of starter leaked magnesium only at pH <3. Those wor- cultures, sensitivity to acid is undesirable be- kers (1 1) suggested that differences in the sus- cause high cell densities may not be achieved, ceptibility to gross membrane damage caused and cell viability may be impaired (77). by acidification appeared to vary among organisms and was correlated with the degree of acid tolerance. ACID DAMAGE DURING GROWTH AT LOW pH Conventionally prepared, milk-based (without added buffers) starter cultures are espe- The basic knowledge that starter cultures cially prone to acid damage because the final grow best at neutral or near neutral pH, that medium pH usually is <5.0. Such cultures, if acid environments are damaging to cells, that they are not used soon, behave poorly in fer- starters tend to lose activity during prolonged mented milks, resulting in slow or sluggish storage at low pH, and that ovempened or culture performance (39, 84). To minimize overacidified cultures perform slowly or result these acid-damaging effects in dairy starter in inferior productions has long been recog- cultures, the starter culture industry has nized (54, 55, 77, 84). Harvey (37) fmt stud- devoted considerable effort to development of

Journal of Dahy Science Vol. 76. No. 8, 1993 2356 HUTKINS AND NANNEN

TABLE 1. Optimal pH for intracellular enzymes from lactic acid bacteria.

~~~~~ PH Enzyme Organism Optimum Reference B-D-Galactosidase Streptococcus thcmphilus 7.1 8-D-Galactosidase S. themphilus 8.0 Phosphofructokinase Lactobacillus bulgaricus 8.2 6-D-Phosphogalactoside Lactococcus lactis galactohydrolase ssp. cremris 7.0 Acylglyccrol Lactococcus lactis ssp. lactis acylhydrolase (lipase) L lactis ssp. cremris 7.0-8.5 Pyruvate kinase L loctis ssp. lactis 6.9-7.5 D-Tagatose 1.6-diphosphate L kactis ssp. lactis aldolase L &tis ssp. cremris 7.0-7.3 Glucose-6-phosphate dehydrogenase Lactobacillus casei 6.8 6-Phosphogluconate de hydrogenase Lb. casei 6.8 Aminopeptidase L lactis ssp. cremoris 7 .O Intracellular proteinase L lactis ssp. lactis 7.5 Aminopeptidase Lb. cmei 6.5 Dipeptidase Lb. casei 7.6 Carboxypeptidase Lb. casei 7.2 X-Prolyl dipeptidyl peptidase L lactis ssp. lactis 8.5 X-hlyl dipeptidyl aminopeptidase L lactis ssp. cremoris 7 .O X-Prolyl dipeptidyl aminopeptidase Lb. bulgancus 6.5 X-Rolyl dipeptidyl aminopeptidase Lactobacillus acidophilus 6.5 H+-ATP=’ s. thcnnoDhilus 7.0-7.5 *Proton-translocatingATPase.

pH-controlled culture propagation systems that ria maintain a neutral cytoplasm. Even prevent low pH in the medium. Several differ- acidophiles, for which the optimal pH for ent means of controlling medium pH have growth may be as low as 2.0, have pHi, near been employed [reviewed by Sandine (105)]. 7.0 (62). Streptococci, lactococci, and other One option is to utilize highly buffered media, lactic acid bacteria generally grow and remain usually containing phosphate or citrate viable within a medium pH range of 4.5 to 7.0 salts (98). One such commercial product, (41). Not surprisingly, therefore, many of the PHASE 4””. releases the buffer components enzymes involved in carbohydrate and amino over time (88). Thunell et al. (99) reported that acid metabolism have pH optima in a neutral PHASE 4”” medium preserved activity and range, as indicated in Table 1. viability of unfrozen cultures for 1 mo at During growth and fermentation, the pH of refrigerated storage. Other systems, called “ex- the medium decreases because of the accumu- ternal pH-controlled systems”, rely on the ad- lation of organic acids, primarily lactic acid. dition of base (usually ammonia or ammonium However, the pH within the cytoplasm of fer- hydroxide) to the starter culture tank (31, 82). menting lactic acid bacteria remains more alka- The optimal pH set for the control of pGutde- line than the medium surrounding the cells pends on the strain of bacteria. (41), largely because the cells rapidly excrete protonated lactic acid, via a carrier-mediated pH HOMEOSTASIS process, into the extracellular medium (30, 51). In addition, the membrane is relatively im- permeable to extracellular protons (and lactate Growth of Bacteria in Acid Environment8 molecules) that are produced during fermenta- In general, microorganisms are able to grow tion. Accordingly, a pH difference between the over a wide pH range from 1.O to 11.O (76). cytoplasm and the medium, a pH gradient Despite this remarkable tolerance, most bacte- (ApH) is formed. The formation and main-

Journal of Dairy Science Vol. 76. No. 8. 1993 SYMPOSIUM: DAIRY STARTER CULTURES 2357 tenance of ApH is important not only for pH 1.5 7- homeostasis but also as a component of the proton motive force (66). The generation and ultimate collapse of ApH in S. fhemphilus is illustrated in Figure 1. A similar pattern also occurs with lactococ- cal cells [Figure 1; (72)l. As pbut decreases from 6.8 to between 5.0 and 5.2, near neutral pHi, is maintained. However, as pH continues to decrease, the cells are apparently unable to maintain ApH; i.e., apparently, at a certain point (p&, 5.0 to 5.2). a large ApH cannot be maintained, ApH begins to collapse, and cell viability is impaired. In contrast, pHi, also decreases in lactobacilli but at a rate that apparently permits the cells to generate and to maintain a large ApH (63, 72). 3 4 5 6 7 8 The pHi, affected growth and numerous External pH metabolic activities in lactic acid and related Figure 1. Formation of a pH gradient during growth of bacteria. In 1978, Harold and van Brunt (36) Streptococcus thennophilus 573 (m) and Lactococcus lactis demonstrated that the maintenance of a neutral ssp. loctis C2 (0) in simulated milk medium. Procedures or slightly alkaline pHi, was required by E. were those discussed by Nannen and Hutkins (72). fuecalis for rapid growth. Similarly, Kobayashi et al. (49) reported that optimal growth of E. fuecalis occurred when the pH of the medium was 6.5 to 7.8, corresponding to pHh 7.5 to cross the cytoplasmic membrane. For example, 7.7. When pHi, was reduced from 7.5 to 6.6, gramicidin D allows exchange of monovalent the growth rate of E. faecalis was reduced by cations (e.g., H+, Na+, and K+), thereby caus- 50% (50). Booth (16) suggested that this bac- ing the ion gradient to collapse. When E. terium could tolerate reductions in pHi, of faecalis was grown in the presence of only 1 pH unit less than its optimum of 7.5. gramicidin (such that pHi, = pHo,J, growth Optimal growth of lactic acid bacteria also occd only when the pH was maintained occurs at near neutral pHi,. Hugenholtz et al. within a narrow range of 7.0 to 7.8 (36). In the (38) determined that the growth rate of L. absence of gramicidin D, E. faecalis was able fuctis ssp. cremoris E8 was maximum at pHoM to grow within a range of pHout4.5 to 9.5 (45). 6.2 (corresponding to an approximate pHi, Those authors (45) concluded that maintenance 7.0). Otto et al. (75) further reported that of neutral pHi, was critical for growth of E. growth of L. lactis ssp. cremoris Wg2 at pHi, fuecalis. Similarly, S. thennophilus lowered 6.7 to 7.0 was independent of pHout (from the pH of Elliker medium to only 6.5 in the pHout 5.7 to 7.0). Ten Brink and Konings (96) presence of gramicidin, whereas control cells showed that pHb of chemostat-grown L lacris (without gramicidin) grew to pH 4.5 (Nannen ssp. cremoris Wg2 remained between 7.5 and and Hutkins, 1991, unpublished data). 6.9 when pbut was varied from 7.5 to 5.5, respectively. Similarly, pbUtof batch-grown Critical pH for Growth of Lactic cells of L lactis ssp. cremoris Wg2 decreased Acid Bacteria from 6.8 to 5.3, and a maximal ApH of .65 pH units occurred at pHout 5.7 (96). The ApH fell Recently, several studies have focused on to zero, however, soon after growth had the actual critical or minimum pHin compatible stopped. for growth of acid-producing organisms. The use of ionophores, such as gramicidin Available literature (8) indicates that such crit- D, have been useful in understanding the role ical pH exist and vary among species. Kashket of pHin on growth and metabolism. These (41) stated that this variation between species substances act by allowing specific ions to is probably due to a slightly different comple-

Journal of Dairy Science Vol. 76, No. 8, 1993 2358 HUTKINS AND NAN" ment of enzymes and transport carriers (41). changes, within tenths of a pH unit, of pHh. In The relatively small amount of data relating the lactic acid bacteria and related species, pHin to cell viability, however, suggests a need nutrient transport and metabolism, protein syn- for greater understanding of acid tolerance in thesis, glycolysis, and nucleic acid synthesis the lactic acid bacteria (16). appear to be regulated by pHh. Some lactic acid bacteria, such as the lactococci and the dairy streptococci, maintain Effect on Ion Transport neutral or near neutral pHi, as pHow decreases as a result of fermentation (78). In contrast, Kashket and Barker (42) showed that, as lactobacilli and ruminant streptococci (i.e,, pHi, of L lacris ssp. luctis decreased, the Streptococcus buvis) are thought to maintain exchange rate of potassium ions for protons high ApH but to allow a reduction of pHi, (85, increased; Le., cells exchanged intracellular 86). For example, Poolman et al. (80) demon- protons for extracellular K+ at low pHh, strated that L lactis ssp. lactis ML3 maintains thereby increasing pHh. At pbut 5.0, an in- pHin around 7.0 even at pHout 5.0. Maloney crease in the ApH (and PHin) by the K+ trans- (58) reported that, at pHout 6.0, L. luctis ssp. port system compensated for decreased elec- luctis maintained ApH of approximately 1.2 trochemical potential. However, at pHout 7.0, pH units (pHin = 7.2). and, at pbut 5.0, ApH the addition of K+ had no affect on the magni- and pHh had increased to approximately 1.9 tude of the ApH. and 6.9, respectively. Kashket et al. (43) also Phosphate ion transport activity in L. luctis reported ApH >1 pH unit (1.1 to 1.4) in grow- ssp. luctis was shown by Poolman et al. (80) to ing and fermenting cells of L lactis ssp. lactis be regulated by pHin. Maximal uptake of phos- at pHout 5.0 to 5.1. Kashket (41) further phate ion in cells grown in lactose occurred at reported that pHi, of Lb. acidophilus, Lb. del- pbutbetween 5.5 and 8.0 (pHi, approximately brueckii ssp. bulguricus, and Lb. cusei 7.2 to 7.5). However, as pHi, was reduced to decreased as pbut decreased. However, cells S6.0, the initial rate of phosphate uptake ap- maintained a large ApH and grew until pHi, proached zero. That inhibition of phosphate around 4.4 was reached. transport by agents that dissipate the ApH McDonald et al. (63) reported similar (e.g., nigericin or carbonyl cyanide m- results for batch-grown cells of Lactobacillus chlorophenylhydrazone) occurred at acidic pH, plantarum, which grew until final pHi, 4.6 to but not at alkaline pH, suggested that phos- 4.8 was reached. In contrast, the less aciduric phate transport activity was regulated by pHi, Leuconostoc mesenteroides responded simi- (80). larly to the lactococci and grew only to pHi, 5.4 to 5.7. Recently, Nannen and Hutkins (72) Effect on Amino Acids reported that the critical pHi, (defined as the and Peptide Transport pHi, at which growth stops and the ApH be- As reviewed recently by Poolman et al. gins to collapse) was 5.0 to 5.5 for L lactis (78), numerous amino acid and peptide trans- ssp. lactis, L. lactis ssp. cremoris, and S. ther- port systems apparently are regulated by pHh. rnophilus. These organisms generally main- van Boven and Konings (100, 101, 102) deter- tained near neutral pHi, as pbutdecreased to mined that peptide hydrolysis and peptide up- <6.0, whereas pHi, in Lb. casei decreased, take were dependent on activities associated even though the latter maintained a large ApH with the cytoplasmic membrane of L. lactis (>1.0), even at low pbut (<4.0). These data ssp. cremoris Wg2. Although the ApH compo- suggest that cessation of growth coincides with nent of the proton motive force appeared to both a low pHi, and the collapse of a large re ulate peptide hydrolysis, uptake of leucyl- ApH. [&]leucine was controlled by pHi, (and, in part, by the ATP pool). When pHin was PHYSIOLOGICAL ROLES OF PHln decreased from 7.3 to 5.4 by addition of nigericin at pbut 6.4, uptake decreased from Somero (91) suggested that significant 16.5 to .9 nmol/min per mg of protein. In changes in the rates of a wide variety of meta- contrast, nigericin had no effect when added to bolic activities were correlated with small cells at pbut7.8 (at pHi, 7.2 to 73, showing

Journal of Dairy Science Vol. 76, No. 8, 1993 SYMPOSIUM: DAIRY STARTER CULTURES 2359 that pHin, and not pHout or the ApH per se, Uptake of DNA determined the rate of uptake of this peptide Recently, Clavd and Trombe (20) suggested (101). that DNA uptake in competent Streptococcus The uptake of amino acids and peptides by pneumoniae cells was strongly dependent on L. lactis ssp. luctis was also studied by Rice et pHh. That bacterium is able to become 100% al. (81). The rate of leucine uptake reached a competent, and, once activated, the uptake of maximum between phUt7.0 and 7.5, and the DNA was not driven by a membrane potential. rate of glycine uptake was maximum at pH However, Clavd and Trombe (20) suggested 6.5. The maximum rate of uptake of dipeptides that DNA transport was dependent on the in- occurred at pH 6.0; however, maximum uptake tracellular ATP concentration and was regu- of tetrapeptides occurred within a larger range lated by PHin. The optimal pHi, for the uptake between pH 6.0 and 8.0. At pHout 4.0, the of DNA was 8.3. uptake of glycyl-leucine by L. lactis ssp. lactis decreased, possibly because of the concomitant Effect on Proteolysis decrease in pHin. De Giori et al. (25) reported that pH had a Dependency on pHi, has also been found strong influence on the proteolytic activity of for the glutamate-glutamine uptake system in lactic acid bacteria in milk systems. The op- L. lads ssp. lactis (79). Those authors (79) timal pH for proteolysis in lactococci was at an reported that the initial rate of glutamate up- external range of pH 5.2 to 5.6, whereas Lb. take increased more than 30-fold when pHi, caei showed high proteolytic activity at pH was raised from 6.0 to 7.4. Driessen et al. (26) 4.8 to 5.2. However, pHin were not inves- examined the effect of pHj,, on L-leucine up- tigated. take in L. lactis ssp. cremoris membrane vesicles and intact cells. In those experiments, the REGULATION OF PHin magnitude of the proton motive force was held The actual means by which cells are able to constant, and pHi, was varied by the use of an maintain constant pHi, despite major fluctua- ionophore, nigericin. A 50% decrease in the tions in the medium pH is an area of intense maximum velocity of L-leucine uptake, from research (16, 46). The relative impermeability .8 to .4 nmoUmin per mg of protein, occurred of the membrane to extracellular protons pro- when ApH was reduced from .6 to 0 at pHout vides cells with some protection and stabilizes 6.0, even though the affinity constant (Kt) was pHb. Despite this physical barrier, however, not affected (26). most organisms have evolved additional Uptake of several amino acids by Lb. cusei mechanisms of controlling and regulating 393 was affected by pHin (93). AS PHin was pHh. Several possible methods have been proposed (16) to control or to maintain pHin op- decreased by the addition of uncouplers or timally, including 1) synthesis or cytoplasmic ionophores to cells at pHout 6.0, transport of buffer, 2) proton symport systems, 3) produc- glutamine, glutamate, and arginine decreased. tion of acids and bases, and 4) proton pumps. No effect was observed by the presence of uncouplers at phut 7.5, indicating that pHi,, Existing Cytoplasmic Buffers not pHout or ApH, regulated transport rates. The results just discussed demonstrate that The cytoplasm of most microorganisms has primary regulation of amino acid and peptide a relatively high buffering capacity. Measure- ment of 50 to 100 nmol of H+ pH unit per mg transport OCCUTS by PHin, not by ApH or pHout (78, 79, 102). Although the activity of some of cell protein have been reported (16) for the buffering capacity of cells at pHin 7.0. Krul- amino acid uptake systems (e.g., those for wich et al. (53) studied the buffering capacity alanine and serine) increases with decreasing of bacilli grown at different pH ranges. The pH, Poolman et al. (78, 79, 80) have made the data suggest that the buffering capacity of the important suggestion that the low uptake rates cytoplasm played little or no role, however, in of essential amino acids at low pHi, (<7.0) ultimately determining the pH range for may account for the growth inhibition of lactic growth of these bacteria. The intracellular acid bacteria that is observed at low pH. buffering capacity may be considered, there-

Journal of Dairy Science Vol. 76, No. 8, 1993 2360 HLJTKINS AND NANNEN fore, to be a limited response used by cells to higher growth rates than when they were counter variations in pHin (16, 34), which sug- grown in conventionally buffered media. gests that other mechanisms are utilized by bacteria. Proton-Translocating ATPase

Proton Symport Systems Although those mechanisms may represent important means to control or to regulate pHh, In 1979, Michels et al. (65) proposed a perhaps the most important homeostatic sys- model of energy recycling to explain how fer- tem in fermentative bacteria is the membrane- menting lactococci could generate a proton bound, proton-translocating ATPase (H+- motive force. In that system, an organism ex- ATPase), which extrudes protons out of the cretes protonated fermentation end products, cell via ATP hydrolysis. This reaction requires such as lactic acid, with a proton:acid energy (in the form of ATP) because expulsion stoichiometry ratio >1; thus, a ApH and proton of protons from a relatively alkaline environ- motive force are generated (51). The continu- ment (i.e., the cytoplasm) into an acidic en- ous production of relatively large quantities of vironment (i.e., the medium) requires move- metabolic end products, coupled to protons, by ment of protons against a concentration fermentative bacteria could contribute signifi- gradient. Although the resulting proton or elec- cantly to a proton motive force and to overall trochemical gradient (also referred to as the production (or conservation) of energy (65, 97) proton motive force) can be used to drive because protons pumps would be spared ATP. uptake of solutes, the main function of H+- In lactococci, the extrusion of protons is ATPase in glycolytic, nonrespiring bacteria thought to be achieved by an electrogenic H+- (Le., lactic acid bacteria) is the maintenance of lactate symporter (41). ApH (34). In most aerobic, respiring microorganisms, this enzyme operates in the opposite Production of Acids and Bases direction to generate ATP. Although ATP synthase activity may also occur in lactic acid The production of acidic or alkaline meta- bacteria (57), under physiological conditions, bolic products by organisms growing in media H+-ATPase functions primarily as a proton at varying pH may be another important means pump (52). of pH regulation (16). For example, synthesis The H+-ATPase from bacteria, as well as of decarboxylases and deaminases may be in- eukaryotic organisms and organelles, have volved in pH regulation. Many lactic acid bac- very similar function and structure. In addition, teria produce these enzymes (e.g., histidine available gene (unc or arp) sequence data have decarboxylase produced by Lactobacillus revealed significant homology. Several reviews buchnerii and arginine deiminase produced by on the structure of bacterial H+-ATPase have L. lactis ssp. luctis). Casiano-Col6n and Mar- recently been published (2, 28, 89, 90, 103), quis (19) reported that ammonia released from and procedures for purification of the enzyme arginine via the arginine deiminase system have been presented (3, 89). Detailed genetic protected lactic acid bacteria against acid dam- studies have also been reviewed (28, 29, 104). age. Marquis et al. (59) suggested that arginol- Stmcture of ATPase. The H+-ATPase com- ysis at low pH occurs as a general adaptive plex consists of two main portions a response by these organisms to acid environ- hydrophilic, peripheral membrane protein, ments. A similar system involving the called F1, and the hydrophobic Fo, which is malolactic system was reported by Daeschel integrated within the membrane (29). In Es- (24). Decarboxylation of the dicarboxylic cherichia coli, the complex has a molecular malic acid and subsequent production of weight of approximately 530,000 Da (90). The monocarboxylic lactic acid by Lb. plantarum F1 is the catalytic portion and is an extrinsic (and other malolactic bacteria) consume an membrane protein consisting of five different intracellular proton and elevate pH. Lac- subunits. The catalytic site of the ATPase is tobacillus plantarum and Leuc. mesenteroides located at the inner surface of the membrane. cells grown in MRS-glucose medium sup- The Fo, a transmembrane complex consisting plemented with malic acid, therefore, achieved of three subunits, mediates the translocation

Journal of Dairy Science Vol. 76, No. 8, 1993 SYMPOSIUM: DAIRY STARTER CULTURES 2361 between two compartments (i.e., the cytoplasm maintain pHi, 7.5. Kobayash (45) suggested and the medium). The passage of the proton that the defect in the acid-sensitive mutant through Fo is, in turn, regulated by the F1 involved the metabolic extrusion of protons portion. Abrams (1) showed that the enzyme through the proton-translocating ATPase and may be detached from the membrane by that the mutants were unable to alkalinize the repeated washings, first with solutions contain- cytoplasm (45). The cytoplasm of the parental ing high salts and then followed by a solution strain was always higher than that of the mu- of low ionic strength and containing no Mg2+. tant, at pH 4.0 (45). Cytoplasmic alkaliniza- Evidence was also presented demonstrating tion by both strains possibly was diminished at that binding of the enzyme to the membrane pHin >7.7 because of reduced proton extrusion was dependent on multivalent cations, such as activity by H+-ATPase. Mg2+, Mn2+, and Ca2+. Abrams and Baron (4) In contrast to effects that occur at high further determined that the addition of Mg2+ pHh, amplification of E. faecalis 9790 H+- ions increased the strength of attachment of the ATPase activity occurred as pHi, decreased enzyme to the membrane but did not increase (48). The addition of gramicidin D-a pro- the total number of binding or catalytic sites. tonophore that allows free diffusion of protons H+-ATPase in Streptococci, Lactococci, across the membrane-to cells grown at pH and Lactobacilli. Harold et al. (35) demon- 7.2 increased H+-ATPase activity fivefold strated that H+-ATPase extrudes protons, from 1.90 to 8.51 unitdmg of protein. Al- resulting in the alkalinization of the cytoplasm though the cells were not able to regulate pHin in E. faecalis. Abrams and Smith (6) further in the presence of protonophores, at pHout showed that the activity of H+-ATPase in E. 4.6, H+-ATPase activity increased. faecalis increased when cells were grown in Abrams and Jensen (5) also examined the K+-limiting medium; .5 mM or greater of K+ altered expression of H+-ATPase in E. faecalis ion was required by E. faecalis to alkalinize in the presence and absence of K+. Results the cytoplasm (47). Some workers (7, 17, 33, showed that H+-ATPase activity in cells grown 36, 42) have suggested that the mechanism in K+-restricted medium (containing .2 mM responsible for raising pHh in enterococci and K+) was more than twofold greater than cells lactococci was the extrusion of protons via the grown in 20.0 mM K+ medium (1.37 vs .59 membrane-bound ATPase combined with the unitdmg of protein). The pH of the medium electrogenic uptake of K+. Kobayashi et al. also affected expression of the enzyme, which (49) confirmed this suggestion by demonstrat- increased from .12 units/mg of protein at pH ing that, as pHi, was shifted to <7.5, H+- 9.0 to .75 units/mg of protein at pH 6.0. ATPase activity in E. faecalis was elevated Research on H+-ATPase activity in E. from 1.8 units/mg of protein at pHh 7.6 (pHout faecalis strongly suggests that this enzyme 7.3) to 3.0 unitdmg of protein at pHi, 7.3 regulates pHin (45, 46, 47, 48, 49, 50). The (pHout6.0). The H+-ATPase activity decreased H+-ATPase activity increases with decreasing when the medium returned to alkaline pH. pH, and, in addition, greater amounts of en- When H+-ATPase activity was inhibited with zyme are produced as pH decreases. Although N’,N’-dicyclohexylcarbodiimide, cells were un- differences exist in the absolute or critical able to keep constant pHh. Kobayashi et al. value at which E. faecalis and lactic acid bac- (49) suggested that the pHi, is regulated by teria regulate their pHin, a similar mechanism changes in H+-ATPase activity, which is, in of pH regulation may exist (16, 41, 46). turn, dependent on pHh. Recently, H+-ATPase from several lactic Mutants that were defective in the regula- acid bacteria have been characterized, and their tion of pHi, were studied by Kobayashi and role in pH homeostasis has been evaluated. Unemoto (50). These acid-sensitive mutants, Niskasaari et al. (74) and Rimpillinen et al. isolated from E. faecalis 9790 (the parental (83) isolated and partially characterized H+- strain), grew at pH 7.5, but not at pH <6.0. At ATPase from the plasma membrane of L. lucfis pHout 6.4, the parent strain maintained a ApH ssp. cremoris. Cells were grown to late log of 1.0 unit, whereas the mutant generated a phase, which decreased the pH of the medium ApH of only .5 unit. The acid-sensitive mutant from 6.7 to 5.2. At pbut 5.2, the specific was unable to generate a ApH large enough to activity of the H+-ATPase was 4.03 pmol of

Journal of Dairy Science Vol. 76, No. 8, 1993 2362 HUTKINS AND NANNEN phosphate releasedlmg of protein per min (83). CONCLUSIONS Given that lactococci grow optimally at near The importance of pH homeostasis and the neutral pH,, high enzyme activity at low pH effects of pHin on metabolic activities in seems to be consistent with research findings microorganisms have become increasingly on E. faecalis. Similar results were reported by recognized. The pHin affected the uptake of Nannen and Hutkins (73); maximum activities nutrients, such as K+, phosphate, and amino occurred at pHout 4.9 to 5.2. However, in the acids. Also, pHi, is an important component of latter study, as pHout decreased below this the proton motive force and, therefore, has a range, H+-ATPase activity declined by 25 to profound effect on the bioenergetic state of the 50%. cell. How pHin is involved or integrated in Several investigators (10, 14, 70, 73) have other metabolic processes and activities, such studied H+-ATPase from lactobacilli. Although as DNA uptake, remains an active and interest- questions related to subunit stoichiometry and ing area for further research. structural arrangements of Lactobacillus H+- The disruption of cytoplasmic membrane ATPase remain unresolved, the activity of the activities in fermentative organisms is as- enzyme is typical to that of other H+-ATPase. sociated with production of acidic fermentation However, some reports (IO, 73) suggest that products. The relative tolerance of these organ- the pH optimum is lower (approximately 5.0) isms to acidic end products is dependent on the than for other lactic acid bacteria, which would strain of bacteria (41). Mechanisms controlling pHin have been the subject of much research, not be unexpected, because Lactobacillus spe- and evidence now indicates that H+-ATPase cies are more tolerant of low pH than lac- plays a key role in the regulation of pHin. This tococci and streptococci; growth of these bac- enzyme directly controlled pHin in E. fuecalis teria in nutrient-rich media may reduce the pH (45, 46, 47, 48, 49). Continued research focus- to as low as 3.0. If acid tolerance is related to ing on H+-ATPase in relation to pH necessary protein-expelling activity, lactobacilli H+- for growth of lactic acid bacteria may improve ATPase would be expected to have a more understanding of the physiology of this impor- acid pH optima than enzymes from other, more tant group of microorganisms. Future studies acid-sensitive bacteria. on the genetic basis of pH homeostasis may Bender and Marquis (10) studied the mem- lead to opportunities for development of acid- brane ATPase and acid tolerance of Lb. cusei. sensitive and acid-resistant lactic starter cul- Only after 4 h at pH 3.0 did damage (deter- tures for specialized applications. mined by leakage of Mg2+) begin to occur to Lb. cusei. Cells that were grown to late ex- ACKNOWLEDGMENTS ponential phase had ATPase activity of 3.29 We thank the National Dairy Promotion and unitslmg of membrane protein (10, 11). By Research Board for its support of this work. comparison, Actinomyces viscosus was also studied by Bender and Marquis (10). That less REFERENCES acid-tolerant strain had H+-ATPase activity of only .6 units/mg of membrane protein. The 1 Abrams, A. 1965. The release of bund adenosine triphosphatase from isolated bacterial membranes differences in acid tolerance between these and the properties of the solubilized enzyme. J. Biol. bacteria may depend on the relative amounts Chern. 240:3675. of ATPase in the cell membranes. 2 Abrams, A. 1985. The proton-translocating membrane ATPase (F~Fo)in Streptococcus faecalis The H+-ATPase of Lb. casei was also stud- Cfaechm). Page 177 in The Enzymes of Biological ied by Nannen and Hutkins (73). Although Membranes. 2nd ed. Vol. 4. Bioenergetics of Elec- activity did not increase with decreasing pH, tmn and Proton Transport. A. N. Martonosi. ed. Plenum Publ. Corp., New York, NY. relatively high basal H+-ATPase activities and 3Abrams, A,, and C. Baron. 1967. Purification and a large ApH were maintained, even when characterization of protoplast membrane ATPase. pHout decreased to <4.0. They (73) suggested Page 163 in Microbial Protoplast, Spheroplasts and that the maintenance of a large ApH at low pH L-Forms. L. Guze, ed. Williams and Wilkins Co., Baltimore, MD. was due to the greater basal production of H+- 4 Abrams. A., and C. Baron. 1968. Reversible attach- ATPase by Lb. casei. ment of adenosine triphosphatase to streptococcal

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Journal of Daily Science Vol. 76, No. 8, 1993