Vol. 144, No. 2JouRNAL OF BACTERIOLOGY, Nov. 1980, p. 672-6820021-9193/80/11-0672/11$02.00/0

Galactose Fermentation by Streptococcus lactis andStreptococcus cremoris: Pathways, Products, and Regulation

TERENCE D. THOMAS,* KEITH W. TURNER, AND VAUGHAN L. CROWNew Zealand Dairy Research Institute, Palmerston North, New Zealand

All of the lactic streptococci examined except Streptococcus lactis ML8 fer-mented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively,in galactose-grown cells compared with glucose- or lactose-grown cells. Reducedintracellular levels of both the lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appearedto be the main factors involved in the diversion of lactate to the other products.S. lactis ML produced only lactate from galactose, apparently due to themaintenance of high intracellular levels of fructose 1,6-diphosphate and triosephosphates. The growth rates of all 10 Streptococcus cremoris strains examineddecreased markedly with galactose concentrations below about 30 mM. Thiseffect appeared to be correlated with uptake predominantly by the low-affinitygalactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined,galactose uptake and initial metabolism involved more extensive use of thehigh-affinity galactose permease and Leloir pathway. With these strains therelative flux of galactose through the alternate pathways would depend on theexogenous galactose concentration.

Lactic streptococci (Streptococcus cremoris,Streptococcus lactis, and Streptococcus diace-tylactis) play a vital role in commercial milkfermentations, where their primary function isto convert lactose to lactic acid (11, 12). Thefermentation of lactose by typical lactic strep-tococci in batch cultures results in both glucoseand galactose moieties being converted simul-taneously and almost entirely to lactate (24, 32).In contrast, the growth of various strains ongalactose results in conversion of only 34 to 74%of the free sugar to lactate (24). Attention wasdrawn to the unusual nature of galactose metab-olism by lactic acid bacteria in the early reviewsof Wiken (33) and Gunsalus (6); although lacticstreptococci have since been shown to produceformate, acetate, and ethanol from galactose (4,13), no fermentation balance has been reportedand the factors responsible for the heterolacticmetabolism observed have yet to be clarified.The lactate dehydrogenase from lactic strep-

tococci has been reported to have an absoluteand apparently specific requirement for fructose1,6-diphosphate (FDP) (see 23). However, it hasalso been shown that tagatose 1,6-diphosphateis an effective substitute for FDP (23, 24) so thatwhichever initial pathway is used for galactosemetabolism (Fig. 1), an activator for lactate de-hydrogenase is present. More recently, LeBlancet al. (13) found that a Lac- mutant of S. lactis

which was also deficient in the galactose phos-photransferase system (PTS) produced moreacetate and ethanol from galactose and con-cluded that the initial pathway used for galac-tose metabolism determined the balance of endproducts. Using continuous cultures to studyend product regulation, Thomas et al. (26)showed that lactic streptococci divert end prod-ucts of either glucose or lactose fermentationfrom lactate to formate, acetate, and ethanolwhen carbohydrate becomes limiting. The phe-notypic change to heterolactic metabolism isaccompanied by a lowering of both the intracel-lular level of FDP and the level of lactate de-hydrogenase, and it was suggested that thesechanges, together with induction of other en-zymes involved in pyruvate metabolism, mayaccount for the products observed. A strikingexception is S. lactis strain ML4, which remainshomolactic under glucose limitation (26).

Lactic streptococci growing on galactose havethe enzymatic potential for galactose metabo-lism via two initially separate routes (2), namely,the D-tagatose 6-phosphate and Leloir pathways(Fig. 1). Operation of the tagatose pathway re-quires translocation of galactose by a phospho-enolpyruvate-dependent PTS and metabolismofgalactose 6-phosphate (Gal-6P) to triose phos-phates via tagatose derivatives. The galactosePTS in S. lactis ATCC 11454 is a low-affinity

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GALACTOSE FERMENTATION BY LACTIC STREPTOCOCCI 673

D - taus - w_o-- GVO_mUp-se

-t

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I ~~~~I\11ALACTOA- -- i t

bYRUVA *A L (O- PLACTAT

. \TA_TOWIAI-WFRLICTONdTPW

FORfATE!

MAD+

MAD+

~~~~~CEATEa I'

FIG. 1. Pathways for galactose metabolism in lac-tic streptococci. Regulatory sites are indicated by A(activation) and I (inhibition). PEP, Phosphoenol-pyruvate.

uptake system with an apparent Km of 15 or 27mM, depending on the growth sugar (13). Beforeentry into the Leloir pathway, free galactose istransported into cells via an ATP-energized, ga-lactose-specific permease system which, in S.lactis ML3, has a similar V. but a 10-fold-higher affinity for its substrate than the galac-tose PTS (30). Although growth rates, enzymelevels, and end products have been measured incertain mutant strains (4, 13), there is no directevidence concerning the relative participation ofthe two pathways in lactic streptococci growingon galactose. The present study was undertakento (i) examine the regulation of end productformation and (ii) determine in growing lacticstreptococci the relative participation of the twoinitial pathways for galactose metabolism.

MATERLALS AND METHODSOrganisms and culture conditions. All strains

were from the collection held at the New ZealandDairy Research Institute. S. lactis 7962 was originallyobtained from the American Type Culture Collection,Rockville, Md.

Unless otherwise specified, static batch cultureswere grown at 300C in T5 broth (27) which contained28 mM galactose and had an initial pH of 7.2. Stan-dardized carbohydrate solutions were filter sterilizedbefore they were added to autoclaved broth (24). Inthis medium, growth stopped at about pH 5.6 due to

galactose exhaustion. For measurements of the rate ofacid production and intracellular levels of enzymesand intermediates, cells were harvested when the re-sidual galactose concentration in the medium was 12to 14 mM (pH, -6.5).Growth rate measurement. Experimental cul-

tures were initiated by inoculating galactose-adaptedgrowing cells into T5 broth with a pH of 6.5. Growthrates were measured turbidimetrically at low cell den-sities and are reported either as doubling times (inminutes) or as specific growth rates (per hour).

Extraction and assay of intermediates. Ex-tracts were prepared from cells growing exponentially,and intermediates were assayed enzymatically with afluorescence spectrophotometer (17, 31, 32). Cells fromeach 25-ml culture (total dry weight of bacteria, ap-proximately 16mg) were collected rapidly by filtrationthrough a membrane filter (diameter, 47 mm; porediameter, 0.8 ,um) and then placed in 5 ml of 0.6 NHC104 at 0°C. The time between the end of filtrationand the immersion of the cells into HC104 was 1 to 2s, and sugar from the growth medium was carried overinto the HC104, indicating that cells did not becomesugar limited during sampling. After 15 min at 0°C,extracts were neutralized with KOH and centrifuged.Supernatants were stored at -70°C until assayed.With these procedures, intermediates (including galac-tose 1-phosphate [Gal-lP]) were stable, and extractionwas maximized.

D-Glyceraldehyde 3-phosphate, dihydroxyacetonephosphate (DHAP), and FDP were assayed by us-ing the glyceraldehyde-3-phosphate dehydrogenasemethod (17). The rabbit muscle FDP aldolase useddid not cleave tagatose 1,6-diphosphate (23). Analysesof Gal-1P and Gal-6P depended upon their markedlydifferent acid labilities (14). After the free galactose inthe extracts was removed by using a column contain-ing a strong base ion-exchange resin (AG 1-X4; Bio-Rad Laboratories, Richmond, Calif.), samples wereheated at 1000C in 0.1 N HCI for 15 min to hydrolyzeGal-1P. After neutralization, the free galactose re-leased was assayed enzymatically by using the proce-dure of Kurz and Wallenfels (10), which was slightlymodified (10 MM NAD+, 50 mM imidazole buffer, pH7.5) for fluorimetric analysis. Treatment of acid-hy-drolyzed samples with alkaline phosphatase allowedthe assay of Gal-6P (29). Similar results were obtainedfor Gal-1P by using the uridyl transferase procedure(37). Experiments with standards showed (i) that therewas complete removal of free galactose and >96%recovery of galactose phosphates when the ion-ex-change column was used and (ii) that acid hydrolysisof Gal-1P was complete, whereas Gal-6P was stable.The intracellular concentrations of intermediates

were calculated on the basis that 1 g (dry weight) ofcells was equivalent to 1.67 ml of intracellular fluid(cytoplasm) (31).Experiments with nongrowing cells. Cultures

growing on galactose were centrifuged, and the cellswere washed and suspended at -2 mg (dry weight) ofbacteria per ml in 20 mM phosphate buffer (pH 6.5)containing 50 mM NaCl and 10 mM MgCl2. The rateof acid production at 30°C and pH 6.50 under nitrogenwas measured with different galactose concentrationsby using a pH-stat apparatus (26).

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674 THOMAS, TURNER, AND CROW

Studies on glucose fermentation by galactose-growncells controlled at pH 6.50 and the extraction of me-tabolites from glycolyzing cells involved proceduresdescribed previously (26).Fermentation product analysis. Cells previously

adapted to grow on the appropriate sugar were inoc-ulated into medium at 30°C containing either galac-tose (16.7 mM), glucose (16.7 mM), or lactose (8.8mM). About 2 h after growth had stopped, culturesamples were centrifuged to remove cells, andL-lactate was measured enzymatically (7). Formate,acetate, and ethanol were assayed by gas-liquid chro-matography (26), and the detection limits for thesecompounds were about 75, 50, and 50 Ag/ml, respec-tively. The absence of residual carbohydrate was con-firmed by enzymatic analysis (24). Uninoculated sam-ples of media were included as blanks for all analyses.H2 production in heterolactic fermentations was ex-amined by growing cultures in sealed tubes and, aftersugar exhaustion, analyzing the air in the headspaceby gas-liquid chromatography.Enzyme assays. Unless indicated otherwise, cells

were disrupted in 20 mM phosphate buffer (pH 6.5)containing 50 mM NaCl and 10 mM MgCl2 by shakingfor 2 min at 0 to 5°C with glass beads in a Mickledisintegrator, and debris was removed by centrifuga-tion at 35,000, x g for 5 min. NAD+-dependent lactatedehydrogenase (EC 1.1.1.27) was assayed immediatelyin supernatant samples by using 0.1 mM NADH andsaturating amounts of pyruvate (10 mM) and FDP (1mM) in 50 mM triethanolamine hydrochloride buffer(pH 6.9). Aldehyde dehydrogenase (EC 1.2.1.10) wasassayed by the method of Stadtman and Burton (20).The alcohol dehydrogenase (EC 1.1.1.1) assay systemcontained 50 mM sodium phosphate buffer (pH 6.7),25 mM acetaldehyde, 0.25 mM NADH, and bacterialextract. A correction for NADH oxidase activity wasmade. Phosphotransacetylase (EC 2.3.1.8) was esti-mated by the method of Stadtman (19), except for thefollowing changes: 20 mM Tris-hydrochloride buffer(pH 8.1), 2 mM acetyl phosphate, 0.05 mM coenzymeA (CoA), 50mM NH4Cl, and 50mM sodium arsenate.Ammonium chloride was essential for activity, whichwas defined as the rate of arsenate-dependent hydrol-ysis of acetyl phosphate. Acetate kinase (EC 2.7.2.1)was assayed by coupling ATP production from acetylphosphate and ADP to NADP+ reduction via hexoki-nase and glucose-6-phosphate dehydrogenase. The re-action mixture contained 100 mM Tris-hydrochloridebuffer (pH 8.5), 5mM MgCl2, 10 mM glucose, 0.5 mMNADP+, 5 mM ADP, 5 U of glucose-6-phosphatedehydrogenase, 12.5 U of hexokinase, 2 mM acetylphosphate, and bacterial extract. Acetyl phosphatewas added last to allow correction for apparent my-okinase activity. The amount of acetyl phosphateused, as estimated by the method of Lipmann andTuttle (16), was equal to the amount of NADPHproduced when corrected for myokinase activity. Allassays were carried out at 25°C, and activity wasproportional to enzyme concentration.The formate-pyruvate exchange activity of pyru-

vate-fornate lyase (EC 2.7.1.40) was assayed anaero-bically in intact and permeabilized cells by using["4C]formate, as described by Wood and O'Kane (34,35) for intact cells. The reaction system contained the

J. BACTERIOL.

following in a total volume of 1.0 ml: 50 ,umol ofpotassium pyruvate, 50 ,umol of sodium ["4C]formate(0.067 ,uCi), 60,imol of potassium phosphate (pH 7.5),0.1 ml of reducing complex (0.01 M FeSO4, 0.03 M 2,3-dimercaptopropanol), and cell suspension. Before thereducing solution and cell suspension were added, thereaction mixture was cleared of oxygen by evacuationand flushing with helium. After incubation for 30, 60,and 90 min at 37°C, reactions were terminated byadding H2S04, and the cells were removed by centrif-ugation. Pyruvate was precipitated by adding 2,4-di-nitrophenylhydrazine, and the derivative was re-covered on a filter paper disk, washed, dried, andcounted in a gas flow counter (model D-47; NuclearChicago Corp.) with an efficiency of 5%. Exchangeactivity increased linearly with time, and the valuesgiven are for a 90-min endpoint. Activity was propor-tional to cell mass, and up to 8% of the total 14C labelwas incorporated into pyruvate during the assay. Aswith the Streptococcus faecalis exchange system (34,35), yeast extract was essential for activity with intactcells, and bubbling 02 into the suspensions causedrapid inactivation. Yeast extract was not required foractivity with permeabilized cells, which were obtainedby adding 1 volume of toluene-acetone (1:9, vol/vol)to 10 volumes of cell suspension (-10 mg [dry weight]of bacteria per ml) in anaerobic buffer (34) at 0°C. At3-min intervals over a 30-min period, the suspensionwas mixed vigorously for 30 a, and cells were thenassayed immediately for exchange activity.Other procedures. Bacterial density was deter-

mined directly by using membrane filters (26). Proteinwas determined by a modification (8) of the Lowrymethod, using bovine serum albumin as the standard.

Materials. All biochemicals were obtained fromSigma Chemical Co., St. Louis, Mo., as the grades withhighest analytical purity. The galactose used con-tained no detectable glucose.

RESULTSGrowth of lactic streptococci on galac-

tose. Doubling times (in minutes) of strainsgrowing at low cell densities with saturatinglevels of galactose (100 mM) were as follows: S.Iactis Clo, 42; S. lactis Hi, 48; S. lactis ML3, 41;S. lactis MLs, 58; S. lactis 7962, 47; S. cremorisAM2, 46; S. cremoris C13, 57; S. cremoris E8, 53;S. cremoris HP, 58; S. cremoris ML1, 66; S.cremoris 104, 68; S. cremoris 108, 56; S. cremoris114, 54; S. cremoris 266, 75; S. cremoris 448, 56;and S. diacetylactis DRCj, 57. During thesemeasurements the galactose concentration inthe medium decreased by -1 mM and the pHdecreased from 6.5 to 6.4, indicating that only asmall shift in the chemical environment oc-curred during these measurements. The pH op-timum for growth of lactic streptococci is usuallyin the range 6.3 to 6.5 (12).

Intracellular concentrations of Gal-1Pand Gal-6P in cells growing on galactose.The intracellular concentrations of Gal-1P andGal-6P, which are intermediates in the Leloir

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GALACTOSE FERMENTATION BY LACTIC STREPTOCOCCI 675

and D-tagatose 6-phosphate pathways, respec-tively (Fig. 1), were measured to indicate whichpathway(s) is actually used for galactose metab-olism. The six strains examined (Table 1) ap-peared to fall into three groups according to therelative levels of the two galactose phosphates.With S. lactis ML3 and 7962, the ratio of intra-cellular Gal-1P concentration to intracellularGal-6P concentration was 2:1, whereas with S.lactis ML8 and S. cremoris AM2 this ratio was1:1. In contrast, S. cremoris E8 andHP contained4 to 6mM Gal-6P but insignificant levels of Gal-ip.The data in Table 1 were obtained when the

galactose concentration in the medium at sam-pling was 12 to 14 mM. In a separate experiment,the intracellular levels of the galactose phos-phates in growing S. lactis ML3 cells were deter-mined as the galactose concentration in the me-dium fell. With the galactose concentration inthe medium at 17.9, 12.7, and 5.9 mM, the levelsof the galactose phosphates were similar tothose shown in Table 1. However, with 3.4 mMresidual galactose in the medium, the intracel-lular Gal-6P concentration decreased to 3.9 mM(intracellular Gal-1P concentration, 11.7 mM),and with 1.5 mM galactose remaining, the intra-cellular levels of both galactose phosphates were-2 mM.The four strains which contained appreciable

levels of Gal-1P (Table 1) also contained 4 to 6mM glucose 6-phosphate when they were grownon galactose. In contrast, S. cremoris strains E8and HP contained no detectable glucose 6-phos-phate, which is consistent with the absence of afunctional Leloir pathway in these strains.Growing on glucose, S. lactis ML3 contained nodetectable (<O.l mM) Gal-1P or Gal-6P. Grow-ing on lactose, S. lactis strains ML3 and MLsand S. cremoris strains EB and HP contained 14to 19 mM Gal-6P, but insignificant levels of Gal-1P, which is consistent with metabolism of thegalactose moiety of the lactose molecule exclu-

TABLE 1. Intracellular Gal-lP and Gal-6Pconcentrations in cells growing on galactosea

Intracellular IntracellularStrain Gal-lP concn Gal-6P concn

(mM) (mM)S. lactis ML3 13.6 + 2.4 7.4 + 1.2S. lactis 7962 12.6 + 2.2 6.1 ± 0.7S. lactis Mb, 6.3 + 1.1 7.3 ± 0.8S. cremoris AM2 5.2 ± 0.5 5.7 ± 1.2S. cremoris E8 0.1 ± 0.1 4.0 ± 0.3S. cremoris HP 0.2 ± 0.2 5.7 + 0.8

aAt sampling, the galactose concentration in themedium was 12 to 14 mM. The results are expressedas means + standard deviations for at least threeseparate experiments.

sively via the D-tagatose 6-phosphate pathway.The presence of an intracellular Gal-6P con-

centration of 6 mM in S. lactis 7962 growing ongalactose (Table 1) suggests that, contrary toearlier reports (9, 12, 31), this strain has a func-tional galactose PTS but that this system doesnot recognize thiomethyl-,8-D-galactopyrano-side.

Effect of galactose concentration in themedium on growth rate. Since the affinitiesof the two alternative uptake systems for galac-tose are markedly different in S. lactis ML3 (30),a study of the effect of the galactose concentra-tion in the medium on the growth rate shouldindicate the relative participations of the twopathways in growing lactic streptococci. Onestrain from each of the three apparent groups(Table 1) was examined (Fig. 2A). With S. lactisML3, the specific growth rate was constant (0.95h-1) as the galactose concentration in the me-dium was decreased from 100 to approximately

10A x * S. Itacis ML

0-8 -

S is ML

2 0-6

I

CD 04-

0-2

10 20 30 40 50

[GALACTOSE]( mM)

FIG. 2. Effect ofgalactose concentration on (A) thespecific growth rate and (B) the rate of acid produc-tion by nongrowing cells. Symbols: 0, S. lactis ML3;0, S. lactis ML8; A, S. cremoris E8. During growthmeasurements in (A), the galactose concentration inthe medium fell by 0.5 to 1 mM and the pH droppedfrom 6.5 to -6.4. With nongrowing cells in a pH-statapparatus (B), the galactose concentration fell by-0.2 mM during measurement.

10 20 30 40Initial [GALACTOSE] medium (mM)

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676 THOMAS, TURNER, AND CROW

10 mM; at lower concentrations the growth ratefell slightly. With S. lactis ML4 and particularlywith S. cremoris E8, the effect of decreasing thegalactose concentration in the medium on thegrowth rate was much more marked. The re-maining 13 strains listed above were also exam-ined. With the exception of strain Mb3, all S.lactis strains and also S. diacetylactis DRC, hadgrowth rates which were virtually unaffected bylowering the galactose concentration in the me-

dium to 2.5 mM. In contrast, the growth ratesof all S. cremoris strains decreased at galactoseconcentrations in the medium of <30 mM untilwith 2.5 mM galactose, the rates were between15 and 50% of the maximum.At the lowest concentration of galactose in the

medium tested (2.5 mM), the concentration de-creased to about 2 mM by the end of growthmeasurement. To establish more precisely theeffect of the galactose concentration in the me-dium on the rate of metabolism, especially withS. lactis ML3, experiments with nongrowingcells were undertaken.Nongrowing cells: effect of galactose

concentration on the iniMtial rate of acidproduction. Galactose-grown cells suspendedin buffer produced acid at rates which weredependent on the galactose concentration andthe strain (Fig. 2B). The initial rate with S.lactis ML3 decreased at <5 mM galactose,whereas the initial rates with S. lactis ML8 andS. cremoris E8 decreased at concentrations of<20 and <40 mM, respectively.End products of galactose metabolism by

growing cells. Metabolism of galactose by lac-tic streptococci was usually heterolactic, withcells diverting various amounts of end product

from lactate to formate, acetate, and ethanol(Table 2). In addition to the data shown, heter-olactic galactose fermentation with productionof formate, acetate, and ethanol was also foundwith S. cremoris AM2, S. lactis C10 and H1, andS. diacetylactis DRCI. The striking exceptionwas S. lactis ML4, which produced only lactatefrom galactose (Table 2). Fermentations of glu-cose and lactose were homolactic for all strainslisted in Table 2, except for lactose metabolismby S. lactis 7962, where formate, acetate, andethanol were also produced. This atypical strainhas a ,B-galactosidase but no phospho-fi-galac-tosidase and appears to take up lactose as thefree sugar (31). Except with S. lactis Mb8, low-ering the initial galactose concentration in themedium resulted in conversion of an even lowerproportion of the sugar to lactate (data notshown). This effect was more pronounced withS. cremoris E8 than with S. lactis ML3. Clearly,the balance of end products may change duringgrowth in batch cultures, and therefore more

detailed studies require control of the galactoseconcentration in the medium and the culturepH, using continuous cultures.The carbon recoveries for the data in Table 2

were >92%, and the oxidation/reduction bal-ances for the heterolactic cultures were <1.0,indicating some missing oxidized product. It ispossible that this material was CO2 produced bythe pyruvate dehydrogenase complex. Partici-pation of this system could also account for theobservation that the sum oftwo carbon products(acetate and ethanol) was greater than formate.CO2 production in heterolactic fermentationswas not determined since lactic streptococci pro-duce C02 from noncarbohydrate substrates. An

TABLE 2. Fermentation products from lactic streptococci growing in batch cultures with different sugars'Amt (mmol) of product per 100 mmol of galactose, 100

Strain Sugar in growth medium mmol of glucose or 50 mmol of lactose fermented"L-Lactate Formate Acetate Ethanol

S. lactis ML3 Galactose 123 47 49 35Glucose 188 NDC ND NDLactose 191 ND 7 4

S. lactis 7962 Galactose 136 44 35 26Glucose 200 ND 7 NDLactose 66 77 83 68

S. lactis Mb5 Galactose 192 ND ND NDGlucose 190 ND ND NDLactose 199 ND ND ND

S. cremoris E8 Galactose 62 97 77 63S. cremoris HP Galactose 61 95 89 60

a Culture conditions were as follows: 30°C; T5 complex medium containing 16.7 mM galactose, 16.7 mMglucose, or 8.8mM lactose; initial pH, 7.2; terminal pH (when sugar exhausted), -6.4 in homolactic cultures and6.1 to 6.2 in heterolactic cultures.

b The carbon recovery for all cultures was >92%.'ND, Not detectable (see text).

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alternative explanation for the observed productratios and oxidation/reduction balances is thatthe organisms contained formate-hydrogenlyase. However, no detectable H2 (<1,0 tmol ofH2 per 100 mmol of galactose fermented) wasproduced in the heterolactic fermentations, thusruling out this possibility.The heterolactic cultures (Table 2) had molar

growth yields which were higher (10 to 20%)than those for homolactic cultures (data notshown), which is consistent with ATP genera-tion during acetate production (Fig. 1).Levels of lactate dehydrogenase, pyru-

vate, and FDP in growing celis. The activityof lactate dehydrogenase in vivo presumably hasan important influence on the end productsformed from pyruvate. Factors affecting this ac-tivity include the actual level of lactate dehydro-genase and the concentrations of substrates andeffector compounds in the cells. The specificactivities of lactate dehydrogenase and the in-tracellular FDP concentrations in S. lactis cells'growing on different sugars are shown in Table3. Compared with homolactic cells, heterolacticcells of S. lactis strains ML3 and 7962 containedreduced levels of lactate dehydrogenase andFDP, especially when strain 7962 was growingon lactose, where conversion of sugar to lactatewas lowest. Although the specific activity oflactate dehydrogenase was also reduced in S.lactis ML8 when it was growing on galactose,these homolactic cells maintained a high intra-cellular FDP concentration, similar to all of thehomolactic cells (Table 3). The intracellularlevel of pyruvate in strain ML3 growing on ga-

lactose was twice that in strains ML8 and 7962(Table 3).The Km values for pyruvate or lactate dehy-

drogenase in cell-free extracts were 1.3 and 2.6mM for strains ML3 and ML5 (galactose grown),respectively, when they were assayed understandard conditions (see above) but with 10mMFDP. Although phosphate (100 mM) produceda slight (<15%) inhibition oflactate dehydrogen-ase activity in vitro, the Km values for pyruvatewere not affected by this compound. Similardata were obtained with extracts from cellsgrown on lactose or glucose.Formate-pyruvate exchange in lactic

streptococci and inhibition by glycolyticintermediates. The production of formate sug-gested the presence of pyruvate-formate lyase,although this system had not been demonstratedpreviously in lactic streptococci. Attempts todetect activity in cell-free extracts were unsuc-cessful. However, using ['4C]formate to assayformate-pyruvate exchange activity in intactcells, we found that lactic streptococci do containthis system at levels which are dependent uponthe sugar used for growth (Table 3). Comparedwith growth on glucose, growth on galactoseresulted in elevated (three- to fourfold) levels ofexchange activity in the three S. lactis strains(including strain ML4) which did not producedetectable formate (Table 2). S. lactis 7962 cellsgrown on lactose, where formate was a majorend product (Table 2), also had elevated ex-change activity (Table 3). An assay for exchangeactivity in S. faecalis ATCC 11700 gave a spe-cific activity value for intact cells comparable to

TABLE 3. Intracellular levels of lactate dehydrogenase, pyruvate-formate lyase, and intermediates involvedin pyruvate metabolism in S. lactis

%Con- ~~~~~~~~~~~~~~~~~~~Intracellu-% Con- Pyruvate- Intracellu- Intracellu- lar D-glyC-S. lactis Sugar in verion Lactate de- formate lar pyru Intracellular lar DHAP eraldehydestrain grohdium to L-lac- sp act"b.c lyase sp vate concn (MM)' concn 3-phos-diumatoeL-lac- sp act ¢ act' (mM)b (MM)b phate concn

(mm)bML3 Galactose 61 9.1 ± 0.7 157 ± 38 6.4 ± 1.0 12.7 ± 0.8 3.7 ± 0.2 0.3 ± 0.1

Glucose 94 19.8 ± 1.6 39 ± 11 18.8 ± 1.4Lactose 95 18.5 ± 2.4 88 ± 26 18.3 ± 1.9

7962 Galactose 68 7.1 ± 0.8 244 ± 76 3.0 ± 0.5 13.0 ± 0.6 3.5 ± 0.1 0.3 ± 0.1Glucose 100 24.8 ± 2.9 72 ± 19 23.1 ± 2.5Lactose 33 83 ± 0.7 217 ± 25 4.8 ± 0.4

ML8 Galactose 96 12.3 ± 2.1 188 ± 44 3.1 ± 0.5 25.0 ± 1.3 7.5 ± 0.4 0.6 ± 0.2Glucose 95 21.9 ± 3.0 56 ± 12 21.2 ± 0.4Lactose 99 21.3 ± 2.2 54 ± 9 19.1 ± 2.1

a From Table 2.b Culture conditions were as follows: 30°C; T5 medium containing 28mM galactose, 28mM glucose, or 15 mM

lactose. Exponentially growing cells were harvested when -60% of the sugar had been fermented. All results aremeans ± standard deviations from at least three independent experiments.

' Micromoles of NADH oxidized per milligram of protein per minute.d Counts per minute exchanged from ['4C]formate to pyruvate per milligram (dry weight) of bacteria under

standard assay conditions.

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678 THOMAS, TURNER, AND CROW

that previously reported (35), and this value wasfive times that obtained for S. lactis ML3 grown

on galactose (Table 3).Using permeabilized cells, we investigated

some properties of pyruvate-formnate lyase. Theapparent Km values for pyruvate were similarfor the enzymes from S. lactis strains ML3 andML4 (6.5 ± 2.5 and 8.1 ± 1.4 mM, respectively).The effects of various glycolytic intermediateson pyruvate-formate lyase activity were inves-tigated since triose phosphates have been re-ported to inhibit the enzyme system from Strep-tococcus mutans (36). With pyruvate concen-trations of 2 and 10 mM, intermediates wereadded to assay systems, and after 30 min, sam-ples were removed for estimation of (i) theamount of 14C exchanged and (ii) the concentra-tion ofthe intermediate added. Addition ofFDP,DHAP, and DL-glyceraldehyde 3-phosphategave inhibition of the exchange reaction (datanot shown), although interconversion of thesecompounds occurred during the assay. Experi-ments with varying initial concentrations of theintermediates indicated that although FDP itselfwas unlikely to be an inhibitor, it appeared thatboth DHAP and glyceraldehyde 3-phosphateinhibited exchange activity. Results for strainsML3 and MLs were similar. Compounds whichhad no effect on exchange activity when theywere added to final concentrations of 10 mMincluded Gal-6P, Gal-1P, glucose 6-phosphate,and fructose 6-phosphate.The intracellular concentrations of triose

phosphates in cells growing on galactose are

shown in Table 3. The levels in strain MLe weretwice those in the other strains, which is con-sistent with the higher intracellular FDP con-centration in strain ML8. Although both triosephosphates appear to inhibit pyruvate-formatelyase, the higher (10-fold) intracellular DHAPconcentration may indicate that DHAP is themore important inhibitor in vivo. The isolationof active pyruvate-formate lyase free from al-

J. BACTERIOL.

dolase and triose phosphate isomerase will berequired for further clarification of the regula-tory properties of this enzyme.

Activities of enzymes involved in acetateand ethanol formation from acetyl-CoA.The activities of these enzymes were measuredto determine whether S. lactis ML4 has theenzymic potential to produce acetate and.ethanol from acetyl-CoA. Table 4 shows thatthe enzymes acetate kinase, alcohol dehydrogen-ase, and phosphotransacetylase were present instrain ML84 as well as in strains ML3 and E8,which did produce acetate and ethanol. Thelevels of some of the enzymes were dependentboth on the strain and on the sugar used forgrowth. Aldehyde dehydrogenase activity (EC1.2.1.10; CoA-dependent) was not detected inany of the three strains grown on galactose or

glucose using either NADH or NADPH. When1 mM NAD+ was substituted for NADH in thestandard alcohol dehydrogenase assay (seeabove), no aldehyde dehydrogenase (EC 1.2.1.3)activity was detected in any of the strains grownon the two sugars.Nongrowing celis: glucose metabolism

by galactose-grown cells. When growing ongalactose, heterolactic cells of S. lactis ML3 and7962 contained lower levels of lactate dehydro-genase and higher levels of formate-pyruvateexchange activity than homolactic cells growingon glucose (Table 3). Galactose-grown cells weretherefore placed in a buffer, and glucose fermen-tation was studied to assess the relative impor-tance of (i) enzyme levels, which remain fixedsince nongrowing S. lactis cells have verylimited ability to synthesize or turn over proteins(25), and (ii) the intracellular concentrations ofeffectors which modulate enzyme activity. Thesenongrowing cells of strains ML3 and 7962 con-

verted 86 and 87%, respectively, of the glucoseto lactate, whereas in growing cultures only 61and 68% of the galactose was fermented to lac-tate (Table 3). Accompanying this change to a

TABLE 4. Activities of enzymes involved in acetate and ethanol formation from acetyl-CoA a

Strain Sugar in growth me- Alcohol dehydrogen- Phosphotrans- Acetate kinase sp act'dium ase sp actb acetylase sp act A

S. lactis ML8 Galactose 0.12 ± 0.05 0.77 ± 0.12 17.5 ± 1.0Glucose 0.03 ± 0.02 0.53 ± 0.25 15.7 ± 1.5

S. lactis ML3 Galactose 0.33 ± 0.14 1.00 ± 0.25 34.0 ± 8.5Glucose 0.25 ± 0.07 0.40 ± 0.09 24.0 ± 5.1

S. cremoris E8 Galactose 0.10 ± 0.02 1.53 ± 0.90 15.3 ± 2.7Glucose 0.03 ± 0.02 0.69 ± 0.08 15.7 ± 2.2

a Culture conditions were as follows: 30°C; T5 medium containing 28 mM galactose or 28 mM glucose.Exponentially growing cells were harvested when -60% of the sugar had been fermented. All results are means± standard deviations from at least three independent experiments.

b Micromoles of NADH oxidized per milligram of protein per minute.Micromoles of acetyl phosphate utilized per milligram of protein per minute.

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more homolactic fermentation was the elevationofthe intracellular FDP concentration to 25 mM(determined when the residual glucose concen-

tration was 2 mM). This elevated FDP leveltherefore stimulated lactate dehydrogenase ac-tivity, and the higher levels of triose phosphatesinhibited pyruvate-formate lyase activity, thusaccounting for the production of more lactate.This is consistent with the observation thatwhereas ML3 and 7962 cells growing on galactoseproduced formate (Table 2), transfer of thesecells to a buffer containing glucose resulted inno detectable formate production and the onlynonlactate end products were acetate andethanol (data not shown). When both strainswere grown on glucose instead of galactose, thenongrowing cells converted -100% of the addedglucose to lactate.The rate of either glucose or lactose fermen-

tation by nongrowing cells remained linear asthe glucose concentrations fell from 10 to <1mM (Thomas, unpublished data). In contrast,the rate of galactose utilization tended to de-crease progressively as the concentration fell.This decrease was apparent with S. lactis ML3but more marked with S. lactis ML8 and espe-cially with S. cremoris E8. These observationsare consistent with the use of a high-affinityuptake system for glucose but a relatively low-affinity uptake system for galactose in the lattertwo strains.

DISCUSSION

Inital pathway(s) for galactose metabo-lism. S. cremoris E8 appears to use predomi-nantly the D-tagatose 6-phosphate pathway forgalactose metabolism (Table 5), as growing cellscontain a high Gal-6P level (-6 mM) and insig-nificant levels of Gal-1P and glucose 6-phos-phate. As high galactose concentrations (>40mM) were required to obtain the maximum ratesof growth and acid production by nongrowingcells of S. cremoris E8, the uptake system (ga-lactose PTS) appears to have a low affinity for

galactose, so that transport is rate limiting with<40 mM galactose. This is in reasonable agree-ment with the data of LeBlanc et al. (13), whodetermined Km values with permeabiized cellsfor the galactose PTS of 15 and 27 mM when S.lactis ATCC 11454 was grown on lactose andgalactose, respectively. A much lower apparentKm value (1 mM) was determined for the galac-tose PTS by using phosphoenolpyruvate-primedcells of S. lactis ML3 (30). This may have re-sulted from strain variation, from the differentmethods used for the Km measurements, or fromthe unusually high intracellular concentration ofphosphoenolpyruvate (11 mM) in these starvedcells (31) compared with cells growing exponen-tially (3).

In contrast to S. cremoris E8, the rates ofgrowth and galactose metabolism in S. lactisML3 were much less dependent on galactoseconcentration. This suggests that strain ML3uses a relatively high-affinity uptake system,presumably the galactose permease since in ML3this system has a 10-fold-greater affinity forgalactose than the PTS (30). This observation,together with the high intracellular Gal-1P con-centration, suggests that at low galactose con-centrations (sufficient to saturate the high- butnot the low-affinity uptake system) the Leloirpathway is the predominant route for galactosemetabolism in S. lactis ML3 (Table 5). However,the participation of the D-tagatose 6-phosphatepathway is likely to increase relative to theLeloir pathway as the galactose concentration inthe medium approaches the level required tosaturate the galactose PTS. This trend would beaccentuated if at a high concentration of galac-tose in the medium the high-affinity system wasrepressed. Except for strain ML8, ML3 was typ-ical of the five S. lactis strains tested; i.e., areduction in the galactose concentration in themedium to 2.5 mM had little or no effect ongrowth rate. In contrast, the growth rate of all10 strains of S. cremoris slowed markedly at a

concentration of galactose in the medium of less

TABLE 5. Participation of Leloir and D-tagatose 6-phosphate pathways in metabolism ofgalactose by S.lactis ML3 and ML8 and S. cremoris E8

Galactose concn (mM) at:Intracelular

Intracellular Gal-6P Pd n ahStrain One-half maxi- One-half maxi- Gal-lP concn

concnPredominant pathwayd

mum specific mum rate of H+ (mM)Y concngrowth ratea productionb (M)

ML3 <1 -0.2 13.6 7.4 LeloirML8 2.5 3.2 6.3 7.3 See textEs 10.2 8.9 -0 5.7 D-Tagatose 6-phosphate

a Derived from Fig. 1A.b Derived from Fig. 1B.'From Table 1 (at sampling, the galactose concentration in the medium was 12 to 14 mM).d May depend on the galactose concentration in the medium (see text).

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680 THOMAS, TURNER, AND CROW

than -30 mM. These results suggest that formost S. lactis strains the predominant initialroute for galactose metabolism at low substrateconcentrations is the Leloir pathway, whereasfor S. cremoris strains the D-tagatose 6-phos-phate pathway is the more important route (ifnot the only route; e.g., in strains E8 and HP).The presence of a galactose permease and theenzymes of the Leloir pathway has not beenestablished in these two strains. With S. lactisMLe, the dependence on the galactose concen-tration in the medium of the growth rate andthe rate of acid production (Fig. 2) suggests theuse of a low-affinity uptake system under theexperimental conditions which we used. How-ever, the kinetic parameters for the galactoseuptake systems in this strain have not beenexamined.The growth rates of typical lactic streptococci

in batch cultures on glucose and lactose and therates of glycolysis by nongrowing cells were max-imal at medium sugar concentrations of 1 mM(Thomas, unpublished data), which is consistentwith the presence of high-affinity uptake sys-tems (phosphoenolpyruvate PTS) for thesesugars (28, 29). Andrews and Lin (1) postulatedthat the "scavenging power" of PTSs is greaterthan that of permease systems, and this wassupported by the growth responses of 12 bacte-rial strains to several ,B-galactosides. However,the present study (see also reference 30) suggeststhat with free galactose the converse situationoccurs in lactic streptococci. Our investigationsprovide an indication of the variability in thesystems initially involved in galactose metabo-lism by lactic streptococci. The flux of galactosethrough the alternate uptake systems and path-ways (Fig. 1) depends not only on their relativeKm values and the galactose concentration in themedium but also on other factors affecting activ-ity and synthesis of these systems.End products from galactose fermenta-

tion. The products from galactose fermentationby lactic streptococci were almost completelyaccounted for as lactate, formate, acetate, andethanol, although different strains producedthese compounds in widely varying proportions(Table 2). This heterolactic fermentation of freegalactose by lactic streptococci may have impli-cations in the development of fermented prod-ucts in which lactase-digested milk is used. Fer-mentation balances for galactose similar to thosein S. lactis strains ML3 and 7962 have beenreported for Streptococcus pyogenes (21). Itseems clear that heterolactic fermentation prod-ucts can arise whichever initial pathway (Fig. 1)is used and that under the experimental condi-tions which we used, fermentation was more

heterolactic with strains which used the low-affinity galactose PTS and D-tagatose 6-phos-phate pathway (i.e., S. cremoris strains). In S.lactis strains, there are exceptions, as galactosefermentation by ML4, apparently via both initialpathways, was homolactic. A Lac- mutant ofstrain ATCC 11454, which was deficient in boththe lactose and galactose PTSs, appeared tometabolize galactose only via the Leloir pathwayand gave a more heterolactic fermentation pat-tern than the parent strain when grown on ga-lactose (13).

Isotope studies have indicated that with S.lactis (4) and S. faecalis (5) the triose phos-phates formed from galactose are metabolizedthrough the glycolytic pathway to pyruvate (Fig.1). Therefore, the actual end products formedare determined by factors regulating the alter-nate routes of pyruvate metabolism. The regu-latory nature of lactate dehydrogenase is wellestablished, with activity dependent on the pres-ence of either FDP or tagatose 1,6-diphosphate(24). In our study, the presence of a highly 02-sensitive pyruvate-formate lyase was demon-strated by using both intact and permeabilizedcells of several strains of lactic streptococci, in-cluding S. lactis strain ML4, which produced nodetectable formate. This enzyme has not beendemonstrated previously in these organisms butis known to be present in citrate-grown S. fae-calis (15) and in glucose-limited S. mutans (36).The products expected from cleavage of pyru-vate by pyruvate-formate lyase are formate inamounts equimolar with acetate plus ethanol,and a balance of the NAD+/NADH pool re-quires that one-half of the acetyl-CoA formed isreduced to ethanol (Fig. 1). Although theamount of ethanol formed (Table 2) was reason-ably consistent with the operation of pyruvate-formate lyase, the amount of formate producedwas less than expected. This may have resultedfrom the production of some acetyl-CoA viaacetaldehyde-TPP by the separate pyruvate de-hydrogenase system (22). The existence of thissystem is supported by the finding that galac-tose-grown cells, when placed in a buffer con-taining glucose, produced acetate and ethanolbut no detectable formate.Three of the enzymes involved in the produc-

tion of acetate and ethanol from acetyl-CoAwere present in each of the lactic streptococciexamined, including S. lactis ML8 (Table 4). Noaldehyde dehydrogenase (CoA-dependent) ac-tivity was detected in the three strains examined(ML8, ML3, and E8), although the fermentationproducts from galactose suggest that this en-zyme is present, at least in strains ML3 and E8.Detection of this enzyme in strains such as ML3

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GALACTOSE FERMENTATION BY LACTIC STREPTOCOCCI 681

and E8 is necessary before the possible absenceof activity in MLs can explain the homolacticfermentation of galactose. Therefore, it is likelythat the end products from galactose metabo-lism are determined by the in vivo activities ofenzymes competing for pyruvate metabolism.Factors affecting these activities include the ac-tual level of enzyme and the concentrations ofsubstrates and effectors inside the celLs. Thelevels of lactate dehydrogenase and pyruvate-formate lyase were decreased and increased, re-spectively, by growing cells on galactose insteadof glucose. However, since these changes werealso observed with the homolactic strain ML,they are not likely to be of major importance. Inaddition, transfer of galactose-grown ML3 and7962 cells to a buffer containing glucose resultedin a more homolactic fermentation and no for-mate production. These observations suggestthat fine control of enzyme activity, rather thancontrol of enzyme synthesis, is largely responsi-ble for the heterolactic fermentation of galac-tose. This fine control appears to be achieved bychanges in the intracellular concentrations ofboth a lactate dehydrogenase activator (FDP)and pyruvate-fornate lyase inhibitors (triosephosphates) (Table 3). It should be noted thatcellular tagatose 1,6-diphosphate levels were notestimated in the present study due to a lack ofthe appropriate aldolase in pure form, so thatthe total intracellular concentration of activatorfor lactate dehydrogenase may have beengreater than indicated by the intracellular con-centration of FDP alone. The intracellular con-centrations of FDP, DHAP, and D-glyceralde-hyde 3-phosphate (Table 3) give equilibriumconstants for FDP aldolase of 8.7 x 10-5 and 8.1X 10-5 M for strains ML3 and 7962, respectively,which are consistent with the value (8.1 x 10-5M) for FDP aldolase from rabbit muscle (18). Incontrast, the value for ML5 is double these val-ues (18 x 10-5 M). This discrepancy could beexplained by a higher tagatose 1,6-diphosphatelevel in Mb3 cells due to a greater participationof the D-tagatose 6-phosphate pathway in thisstrain. It is not clear why ML* cells growing ongalactose contain levels of effector compoundswhich are twice the levels in the other twoheterolactic strains. However, these high intra-cellular concentrations (25 mM FDP, 7.5 mMDHAP, and 0.6 mM glyceraldehyde 3-phos-phate) appear to be of overriding importance incontrolling pyruvate metabolism. Similar con-clusions were reached with glucose-limitedchemostat cultures of lactic streptococci, wherelower intracellular levels of FDP (and presum-ably triose phosphates) corresponded to a switchto heterolactic fermentation, whereas ML8 re-

tained high intracellular FDP concentrations (24to 29 mM) and remained homolactic (26). Otherfactors likely to influence pyruvate metabolisminclude the different affinities of lactate dehy-drogenase and pyruvate-formate lyase for theirsubstrates and variations in their intracellularconcentrations. Therefore, the intracellular lev-els of the common substrate (pyruvate) and theaffinities of some of the enzymes competing forpyruvate metabolism were measured. Pyruvate-formate lyase had a lower affinity for pyruvate(Ki, 6 to 8 mM, compared with a Km of 1 mMfor lactate dehydrogenase) and is therefore moreactive in cells with high intracellular pyruvateconcentrations (e.g., MLb [Table 3]).When lactose is fermented by lactic strepto-

cocci (except the atypical strain 7962), the galac-tose moiety (in contrast to free exogenous galac-tose) is fermented almost entirely to lactate.This appears to result from the relatively highlevels of FDP and lactate dehydrogenase (Table3) and the presumed accompanying high levelsof triose phosphates (and reduced levels of py-ruvate-formate lyase); i.e., cells growing on lac-tose contain levels of the factors affecting endproduct regulation which are very similar tothose in cells growing on glucose (Table 3).The present study clarifies some of the factors

regulating end product formation in lactic strep-tococci. Conditions which lead to reduced intra-cellular FDP concentrations (e.g., galactose me-tabolism or glucose limitation) diminish lactatedehydrogenase activation, and the reduced in-tracellular triose phosphate concentrations re-lease pyruvate-fornate lyase inhibition and di-vert product from lactate to fornate, acetate,and ethanol. In addition, it is apparent that thechanging galactose concentration in the mediumduring batch culture experiments not only re-sults in altered growth rates but also is likely tochange the relative participation of the two ini-tial pathways, the intracelular concentrations ofregulatory metabolites, and the actual balanceof end products. Clearly, an examination of arange of strains will be required for an adequateunderstanding of galactose metabolism in lacticstreptococci, and further studies would be as-sisted by the use of continuous cultures to con-trol the galactose concentration in the medium.

ACKNOWLEDGMENTS

We thank Judith Cleland for excellent technical assistance,A. M. Roberton, Auckland University, for help with anaerobicprocedures for the pyruvate-formate lyase assay, and R. C.Lawrence for helpful discussions.

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