JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 76-83 Vol. 153, No. 10021-9193/83/010076-08$02.00/0Copyright © 1983, American Society for Microbiology

Plasmid Linkage of the D-Tagatose 6-Phosphate Pathway inStreptococcus lactis: Effect on Lactose and Galactose

MetabolismVAUGHAN L. CROW,* GRAHAM P. DAVEY, LINDSAY E. PEARCE, AND TERENCE D. THOMAS

New Zealand Dairy Research Institute, Palmerston North, New Zealand

Received 12 July 1982/Accepted 12 September 1982

The three enzymes of the D-tagatose 6-phosphate pathway (galactose6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphos-phate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococ-cus lactis Clo, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-p-galactosidase activitieswere also absent in Lac- derivatives of strains H1 and 133 and were low (possiblyabsent) in C1o Lac-. In all three Lac- derivatives, low galactose phosphotransfer-ase system activity was found. On galactose, Lac- derivatives grew more slowly(presumably using the Leloir pathway) than the wild-type strains and accumulatedhigh intracellular concentrations of galactose 6-phosphate (up to 49 mM); nointracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lacphenotype is plasmid linked in the three strains studied, with the evidence beingmore substantial for strain H1. A Lac' derivative of H1 contained a single plasmid(33 megadaltons) which was absent from the Lac- mutant. We suggest that thegenes linked to the lactose plasmid in S. lactis are more numerous than previouslyenvisaged, coding for all of the enzymes involved in lactose metabolism frominitial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway.

Lactic streptococci (Streptococcus cremorisand S. lactis) transport lactose via a phos-phoenolpyruvate (PEP)-dependent phospho-transferase system (PTS) (26, 37). The lactosephosphate formed is hydrolyzed by phospho-f3-galactosidase (16), giving D-glucose and D-galac-tose 6-phosphate (Gal-6P) (37). The latter inter-mediate is further metabolized to triosephosphates by the three enzymes (see Fig. 2) ofthe D-tagatose 6-phosphate (Tag-6P) pathway(3).

In Staphylococcus aureus, the Tag-6P path-way is required for utilization of galactose aswell as lactose (4). Lactic streptococci, howev-er, have the enzymatic potential to metabolizegalactose via two initially separate routes, name-ly, the Tag-6P pathway and the galactose 1-phosphate (Gal-1P) pathway (Leloir pathway)(3). Either one or both pathways appear tooperate, depending on the strain and the exoge-nous galactose concentration (36). With most S.lactis and S. cremoris strains, galactose fermen-tation is heterolactic (36) whereas lactose fer-mentation is homolactic (33).The instability of lactose metabolism in lactic

streptococci was noted by earlier workers (14,15, 40), and the frequency of lactose-negative

(Lac-) variants was increased by curing agents(24, 25, 27). Subsequent work (for a review, seereference 10) has supported the early suggestion(25) that lactose metabolism is plasmid linked.The lactose-PTS and phospho-,B-galactosidasehave been associated with the lactose plasmid instrains of S. lactis and S. cremoris (1, 20, 21, 26,32), and the lactose plasmid influences the me-tabolism of galactose by S. lactis (21). However,the three enzymes involved in the metabolism ofGal-6P (see Fig. 2) have not been assayed inLac- variants. A deficiency in one or more ofthe three Tag-6P pathway enzymes could resultin a Lac- phenotype.The present data suggest that all three Tag-6P

pathway enzymes are associated with the lac-tose plasmid in S. lactis. In addition, we investi-gated the influence of this association on themetabolism of galactose in Lac- strains of S.lactis.

MATERIALS AND METHODS

Organism and culture conditions. S. lactis strainswere from the collection held at the New ZealandDairy Research Institute, Palmerston North, and arelisted in Table 1.

Static batch cultures were grown at 300C in T5

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PLASMID-LINKED LACTOSE METABOLISM IN S. LACTIS 77

TABLE 1. Strains of S. lactis

Designation phent Denvation Fq,,n,y Plasmid(s) (Mdal)phenotype Deiaio%

H1 Lac+ Wild type (forms clumps in broth) 54, 33, 21, 3.74027 Lac+ Nonclumping <0.01 33, 214122 Lac+ From 4027 after curing at 38.5°C 25 334125 Lac- Gald From 4122 (spontaneous derivative) 0.5 None

C10 Lac+ Wild type a4046 Lac- Gald From Clo after curing at 40°C 18

133 Lac+ Wild type received as NCDO 496 (ATCC 11454and DR1251 [21])

4070 Lac- Gald From 133 after curing at 40°C 8.9a _, Consistent differences in plasmid profiles between Lac+ and Lac- Gald strains were not attainable.

complex broth (35) which contained 28 mM galactose,28 mM glucose, or 14 mM lactose, and the initial pHwas 7.2. Standardized carbohydrate solutions werefilter sterilized before addition to autoclaved broth.Cells were grown for at least 20 generations on theappropriate carbohydrate before use in experiments.

Plasmid curing. Lactose-defective derivatives of S.lactis C1O and 133 were obtained by growing culturesat -40°C for 4 to 6 h in glucose-M17 broth (9). Afterexposure to curing temperature, cultures were platedon SALT medium and incubated at 30°C for 48 h. TheSALT medium contained 0.5% peptone (BBL Micro-biology Systems, Cockeysville, Md.), 0.1% Trypticase(BBL), 0.1% tryptone (BBL), 0.25% yeast extract(BBL), 1.9%o disodium P-glycerophosphate (grade II;Sigma Chemical Co., St. Louis, Mo.), and 1% agar.After adjusting the pH to 6.8 and autoclaving, weadded sterile L-arginine hydrochloride, lactose, andtriphenyltetrazolium chloride solutions to final con-centrations of0.4, 0.5, and 0.005%, respectively. Lac-derivatives were purified from small-colony isolates.Plasmid analysis. Plasmid DNA was prepared after

lysis by the method of LeBlanc and Lee (22). Electro-phoresis was carried out in horizontal 0.55% agarosegels.Enzyme assays. Cells growing exponentially were

harvested when the residual carbohydrate concentra-tion in the medium was half the initial concentration(pH -6.5). Cells were disrupted in 20 mM phosphatebuffer (pH 6.5) containing 50 mM NaCl, 10mM MgCl2,and 1 mM dithioerythritol by shaking for 2 min at 0 to5°C with glass beads in a Mickle disintegrator. Debriswas removed by centrifugation at 35,000 x g for 5 min.Cell extracts were stored on ice, and enzyme assayswere completed within 2 h from preparation of ex-tracts. All enzymes assayed were stable (>90%) for atleast 6 h. For the cell extracts in which no enzymeactivities were detected, at least two further extractswere prepared, and the appropriate enzyme activitieswere assayed immediately.

D-Tagatose 1,6-diphosphate (TDP) and D-fructose1,6-diphosphate (FDP) aldolases were assayed as de-scribed previously (8). The standard assay for D-fructose 6-phosphate (Fru-6P) kinase and Tag-6P ki-nase contained (in a 1-ml volume) 50 mMtriethanolamine hydrochloride buffer (pH 7.8), 0.25mM NADH, 2 mM ATP, 7 mM MgCl2, nonlimitingamounts of the coupling enzymes a-glycerophosphate

dehydrogenase (1.2 U), triosephosphate isomerase(11.5 U), either rabbit muscle FDP aldolase (1.2 U) orTDP aldolase (0.6 U, purified from S. cremoris E8 [8]),limiting amounts of kinase, and either 10 mM Fru-6Por 2 mM Tag-6P. The reaction was followed at 340 nm(25C) with a spectrophotometer (model 250; GilfordInstrument Laboratories, Inc., Oberlin, Ohio). Cor-rections for NADH oxidase activity were made. Nointerfering dehydrogenase activities were observed,and the reaction rate was proportional to kinase con-centration. One unit of kinase activity was defined asthe amount of enzyme that catalyzed the phosphoryla-tion of ketohexose phosphate at an initial rate of 1,umol/min. Gal-6P isomerase was assayed by a three-step procedure. In step 1, the isomerase was incubatedin an assay mixture (100 ,ul) containing 100 mMtriethanolamine hydrochloride buffer (pH 7.8) and 10mM Tag-6P for 0, 15, and 30 min. The reaction wasstopped by heating in a boiling-water bath for 5 min. Instep 2, 30 ,ul of 1 M glycine-NaOH buffer (pH 10.5) and70 ILI of alkaline phosphatase (7 U) were added to theheat-treated reaction mixture, followed by incubationfor 60 min at 25C. Step 3 involved the enzymaticdetermination of galactose (19). One unit of isomeraseactivity was defined as the amount of enzyme thatcatalyzed the formation of Gal-6P from Tag-6P at aninitial rate of 1 ,mol/min. ,-Galactosidase and phos-pho-,-galactosidase activities were assayed in a 1-mlreaction mixture containing 50 mM sodium-potassiumphosphate buffer (pH 7.2), either 1 mM o-nitrophenyl-,-D-galactopyranoside (ONPG) or 1 mM ONPG-6-phosphate (ONPG-6P), and limiting amounts of either3-galactosidase or phospho-o-galactosidase. The reac-

tion was followed at 410 nm and 25°C. One unit ofphospho-p-galactosidase or ,-galactosidase was de-fined as the amount ofenzyme that hydrolyzed 1 pmolof ONPG-6P or ONPG, respectively, per min.PTS activities were assayed in permeabilized cells

which were prepared by a modification of the methoddescribed by LeBlanc et al. (21). The modificationinvolved the use of a different buffer (20 mM phos-phate buffer [pH 6.5] containing 50 mM NaCl, 10 mMMgCI2, and 1 mM dithioerythritol) and 100 p.1 oftoluene-acetone (1:9) per ml of cell suspension. ThePTS assay mixture contained the following (total vol-ume, 1 ml): 5 mM PEP, 10 mM NaF, 0.1 mM NADH,13 U of rabbit muscle lactate dehydrogenase, 50 mMsodium-potassium phosphate buffer (pH 7.2), 5 mM

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78 CROW ET AL.

MgCl2, 50 ,il of permeabilized cell suspension, andcarbohydrate substrate added to a final concentrationof either 10 mM glucose, 5.0 mM lactose, or 40 mMgalactose. The rate of NADH oxidation was followedat 340 nm and 25°C. NADH oxidase controls consistedof the assay mixture without added PEP. Controlswith PEP but no added carbohydrate were also rou-tinely done. Specific activities are expressed as nano-moles of NADH oxidized (PEP and carbohydratedependent) per minute per milligram (dry weight) ofcells. P-Galactosidase and phospho-p-galactosidaseactivities were assayed in permeabilized cells with thesame reaction mixture as described above for cellextracts.

Extraction and assay of intermediates. Cells growingexponentially were extracted with perchloric acid, andthe intermediates were assayed enzymatically in neu-tralized extracts with a fluorescence spectrophotome-ter (23, 36). FDP and TDP were assayed by a glyceral-dehyde 3-phosphate method (8); the Gal-lP and Gal-6Pmethods used have also been described previously(36).Other procedures. Fermentation product analysis

and growth rate measurements were as describedpreviously by Thomas et al. (36). Protein was deter-mined by a modification (12) of the Lowry method,using bovine serum albumin as the standard. Thebacterial dry weight of permeabilized cells was deter-mined directly with membrane filters (34).

Materials. All biochemicals (of the grade with high-est analytical purity) were obtained from Sigma Chem-ical Co.

RESULTS

Lac- variants: Isolation and growth. By use ofelevated growth temperatures, Lac- variantswere readily obtained for strains 133 and C1o.Spontaneous Lac- variants of the single-plas-mid-containing H1 derivative (4122) were isolat-ed (Table 1).T5 complex broth supported some growth

without addition of carbohydrates (maximumcell density, -0.08 mg [dry weight] per ml,compared with 1.07 mg [dry weight] per ml with14 mM lactose). The Lac- strains showed nofurther growth upon addition of lactose, and nodetectable lactose utilization occurred during 48h of incubation. Previous reports indicated thatthe Lac- strains are able to grow on galactosebut with longer generation times (11, 21, 28) anda shift to a more heterolactic fermentation (11,21). These mutants have been designated Lac-Gald (28). The strains studied by us showedsimilar properties. Compared with the Lac'strains of C1o, H1, and 133, the doubling timesfor the respective Lac- Gald derivatives (initialgalactose concentration, 20 mM) increased from53 to 78 min, 51 to 70 min, and 65 to 87 min, withan associated decrease in the percent conversionof galactose to L-lactate (54 to 23%, 51 to 30/%o,and 51 to 29%, respectively). No detectablegalactose (<0.5 mM) was present at the end ofgrowth when the lactate measurements were

made (initial galactose concentration, 28 mM).All six cultures, independent of the Lac pheno-type, converted >95% of glucose to lactate.

Plasmid analysis. The plasmid profiles ofstrain HI and its derivatives obtained by curingare shown in Fig. 1. The nonclumping strain4027 had lost both the 54- and the 3.7-megadal-ton (Mdal) plasmids. Strain 4122 had further lostthe 21-Mdal plasmid but retained a single plas-mid (33 Mdal) and with it the Lac' phenotype.The spontaneous Lac- Gald strain 4125 wasplasmid free. Repeated plasmid extractions ofthe Lac- Gal" derivatives from strains 133 andClo run on gels showed no consistent differencesfrom the wild types.Tag-6P pathway enzyme activities. The first

enzyme of the Tag-6P pathway (Gal-6P isomer-ase) was not found in any of the three Lac Galdderivatives, whereas considerable activity wasdetected in the three Lac' strains (Table 2). Thesecond enzyme (Tag-6P kinase) was present inthe Lac- Gald mutants but at specific activitieswhich were 20 to 36% of those in the Lac'strains. The last enzyme of the Tag-6P pathway(TDP aldolase) was detected in the three Lac'strains but not in the Lac- Gald derivatives(Table 2). In contrast to Tag-6P kinase, Fru-6Pkinase activity was not significantly differentbetween the Lac- Gald and Lac' cultures. FDP

A B C D

54

FIG. 1. Agarose gel electrophoresis patters ofplasmid DNA isolated from S. lactisH, and deriva-tives. (A) Hl, (B) 4027, (C) 4122, and (D) 4125.Molecular weights (X106) were determined from ly-sates containing E. coli plasmids of known size. chr,Chromosome.

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PLASMID-LINKED LACTOSE METABOLISM IN S. LACTIS 79

TABLE 2. Specific activities of enzymes involved in lactose metabolism in wild-type and Lac- Gallderivatives of S. lactis C1o, H1, and 133 grown on galactosea

Enzyme sp actb

Enzyme C0- HI 133Lac' Lac- Gald Lac+ Lac- Gald Lac+ Lac- Gald

Gal-6P isomerase 4.30 NDc 2.80 ND 1.10 NDTag-6P kinase 0.83 0.17 0.50 0.18 0.53 0.11TDP aldolase 0.51 ND 1.03 ND 0.53 NDFru-6P kinase 0.77 0.96 0.93 0.94 0.77 0.88FDP aldolase 3.29 1.77 3.16 2.68 2.56 1.67Phospho-,-galactosidase 0.80 0.02 0.22 ND 0.97 ND,B-Galactosidase 0.004 0.04 ND ND ND ND

a Culture conditions, preparation of cell extracts, and enzyme assay procedures are described in the text.b Specific activities are expressed as micromoles of substrate utilized per milligram of protein per minute.c ND, Not detectable.

aldolase activity was detected in all six cultures,although the Lac- Gald derivatives had a loweractivity than their respective Lac' strains. Thislower activity is explained by the absence ofTDP aldolase (Table 2), which is active withboth FDP and TDP (8). No phospho-3-galacto-sidase activity could be detected in the Lac-Gald derivatives of H1 and 133, whereas slightactivity (2.5% of that of the parent strain) wasdetected in the Lac- Gald derivative of C1O. LowP-galactosidase activity was detected in theLac' and Lac- Gald cultures of C1o, whereas noactivity of this enzyme was found in the othertwo strains or their variants.The Fru-6P and Tag-6P kinases from S. lactis

Clo have been separated and their propertiesstudied (A. M. Fordyce, Ph.D. thesis, MasseyUniversity, Palmerston North, New Zealand,1982). Fru-6P kinase is an allosteric enzymewith some activity for Tag-6P (half-maximalvelocity for Tag-6P [Tag-6P0.5 v], 2.82 mM). Incontrast, Tag-6P kinase is a nonallosteric en-zyme with a high affinity for Tag-6P (Km = 0.16mM). It is therefore possible that the apparentTag-6P kinase activities found in the Lac- Galdderivatives (Table 2) were in fact due to Fru-6Pkinase activity. To examine this possibility, thekinase kinetics for Tag-6P in crude cell extractsfrom C1o and its Lac- Gald derivative werestudied. For C1o, where both enzymes are pres-ent, the Lineweaver-Burk plot was biphasic,with one slope (in the region of 0.1 to 1.0 mMTag-6P) extrapolating to give a Km value of 0.07mM Tag-6P. From the same plot, with data from1.0 to 5 mM Tag-6P, a much higher Km value(approximately 10 mM Tag-6P) was obtained. Incontrast, for the Lac- Gal derivative of C1o, theLineweaver-Burk plot indicated that only a low-affinity system was present for Tag-6P. As thekinetics indicated cooperative binding, a Hillplot was used to determine the Tag-6P concen-tration giving half-maximal velocity (Tag-6PO.5 v

= 6.4 mM) and the Hill interaction coefficient(1.8). Therefore, it is possible that in the Lac-Gald derivative of C10 the activity with Tag-6Pwas due only to the activity of Fru-6P kinase.PTS activities in Lac+ and Lac- Gald strains

grown on galactose. No lactose-PTS activity wasdetected in the Lac- Gald derivatives of H1 and133, whereas C10 Lac- Gald had -5% of thelactose-PTS activity of the parent strain (Table3). We considered the possibility that this lowlactose-PTS specific activity was an artifact.Contamination of the lactose substrate with glu-cose (<0.01%) could not explain the low lactose-PTS activity in C10 Lac- Gald. This Lac- Galdderivative had low ,-galactosidase activity incell extracts (Table 2) and permeabilized cells(16 nmol of ONPG released per mg [dry weight]of bacteria per min) which could supply glucosesubstrate for the high-affinity glucose-PTS (Km= 0.02 and 0.04 mM glucose for 133 and C10,respectively). However, incubating permeabi-lized cells of C10 Lac- Gald with lactose fordifferent times before addition of PEP did notalter lactose-PTS activity. This suggested thatPTS activity is associated with lactose ratherthan with any glucose produced by 3-galacto-sidase. The lactose-PTS showed a high affinityfor lactose (Km = 0.07 and 0.03 mM lactose forC10 and 133, respectively).

In all three Lac- Gal derivatives, the galac-tose-PTS specific activity was much lower thanin the parent strains (Table 3). This activity inthe Lac- Gald derivatives was galactose specificand not due to glucose contamination (<0.01%)of the galactose. For strain 133 and its Lac- Galdderivative, the Km for galactose was 20 mM,suggesting that the same low-affinity PTS forgalactose was present in both. In strains Cl0 andH1, the galactose-PTS had Km values of 18 and 7mM, respectively. Thus, relative to the affinitiesfor glucose and lactose, the affinity of the PTSfor galactose was low in the three strains.

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TABLE 3. Specific activities of phosphotransferase systems in permeabilized cells of wild-type and Lac-Gald derivatives of S. lactis C1o, H1, and 133 grown on galactosea

Sugar-PTS sp actbPTS CI0 H1 133

Lc+ LacGald Lac Lac Gaid L+ Lac- GaId

Lactose 138.3 6.4 7.3 NDC 52.0 NDGalactose 64.2 2.1 5.6 1.2 19.7 5.6Glucose 117.6 19.9 7.9 3.5 46.1 34.0

a Culture conditions, preparation of permeabilized cells, and enzyme assay procedures are described in thetext.

b Specific activities are expressed as nanomoles of NADH oxidized (PEP and carbohydrate dependent) perminute per milligram (dry weight) of cells.

c ND, Not detectable.

P1TS specific activities for all three carbohy-drates were low for H, compared with thosefound for 133 and C1O (Table 3). The use ofdifferent procedures for permeabilizing cells didnot increase this activity. Although strain H,cells aggregate to form large clumps, PTS activi-ties were still low in a nonclumping variant ofthis strain (4122; Table 1). Nongrowing S. lactisCl0 cells metabolize carbohydrate (20 mM) atrates of 81, 89, and 43 nmol of carbohydrate(respectively, galactose, glucose, and lactose)per mg (dry weight) of bacteria per min (7).These values were similar to the respective PTSspecific activities (Table 3) for C10.

Intracellular concentrations ofsome key metab-olites in galactose-grown cells. The intracellularconcentrations of Gal-6P in the Lac- Gald deriv-atives were markedly elevated (4- to 14-fold)compared with those in the parent strains (Table4). There was no detectable TDP (and presum-ably no Tag-6P) in the Lac- Gald cells (Table 4).

All Lac' strains contained both Gal-6P andGal-1P, consistent with galactose metabolismvia both the Tag-6P pathway and the Leloirpathway (36). Previous work (8, 34, 36) estab-lished that fermentation of lactic streptococcibecomes more heterolactic when the intracellu-lar concentrations of FDP and TDP decrease.The three Lac- Gald derivatives, which, asmentioned previously, were more heterolacticthan their respective parent Lac' strains, alsohad decreased intracellular concentrations ofFDP (Table 4).

DISCUSSIONAlthough most lactic streptococci carry a

number of different plasmids, there has beenconsiderable difficulty in attributing these plas-mids to specific phenotypes. Strains defective inlactose metabolism can often be isolated at highfrequency either spontaneously, after growth atan elevated temperature, or with known curingagents. The plasmid profiles of such Lac-strains may or may not show the absence of

plasmid bands, and these strains are sometimesdefective in other phenotypes (e.g., proteinase).The accumulated evidence from several investi-gators suggests that in a number of lactic strep-tococci the specific components of the lactose-PTS and phospho-3-galactosidase are plasmidencoded (for a review, see reference 10). Genet-ic transfer with plasmid-free recipients has insome instances clarified the role of specificplasmids. In a plasmid-free S. lactis C2 recipi-ent, Lac' transconjugants containing a singleplasmid were obtained with an S. lactis ML3donor (39). With S. cremoris donors and thesame recipient, however, Lac' transconjugantscontained one or more plasmids (31).Because of the difficulties encountered in the

interpretation of the results for many lacticstreptococcal systems, the choice of strains usedfor detailed study is clearly of importance. Thetwo S. lactis H1 derivatives best fulfill the

TABLE 4. Intracellular concentrations of someintermediates in S. lactis strains and their Lac- Gald

derivatives growing on galactoseaIntracellular concn (mM)

StrainGal-6P TDP Gal-IP FDP

c1oLac+ 3.0 2.1 3.3 17.0Lac-Gald 13.2 NDb 2.0 13.2

H1Lac+ 3.7 0.8 6.3 10.8Lac- Gald 27.2 ND 5.0 5.9

133Lac+ 3.5 1.9 9.7 20.0Lac- Gald 49.4 ND 5.9 12.3a Culture conditions and measurements of intracel-

lular intermediate concentrations are described in thetext. Mean values from at least three separate experi-ments are given. At sampling, the galactose concentra-tion in the medium was 12 to 14 mM.

b ND, Not detectable.

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PLASMID-LINKED LACTOSE METABOLISM IN S. LACTIS 81

criteria for unambiguous plasmid analysis. Thesole plasmid (33 Mdal) in a Lac' derivative ofH1 (4122) was not detected in the Lac- Galdderivative (4125). It seems likely that loss of theLac phenotype was associated with loss of thissingle plasmid since (i) Lac- Gald mutants arosespontaneously at high frequency (0.5%), (ii) re-version to Lac' has never been observed, and(iii) up to six enzymes were lost. Plasmid pro-files of the Lac- Gald derivatives of Cl0 and 133showed no consistent differences from the wildtypes. However, it is likely that a missing plas-mid does account for the Lac- Gald phenotypeobserved in these derivatives because of theirsimilarities with the other above-mentioned prop-erties of H1 Lac- Gald. Other workers, usingdensity gradient purification, have shown dele-tion of a 32-Mdal plasmid in an independentlyderived Lac- Gald variant of 133 (21).The specific activities of the three Tag-6P

pathway enzymes in three strains growing ongalactose were markedly influenced by the Lac-Gald phenotype (Table 2). Previous studies havenot investigated these enzymes in Lac- Galdphenotypes of lactic streptococci. Gal-6P isom-erase and TDP aldolase activities were not de-tectable in the three Lac- Gald strains, whereasthe third enzyme (Tag-6P kinase) showed adecreased specific activity. As the Fru-6P kinasein Escherichia coli (2) and S. lactis (A. M.Fordyce, Ph.D. thesis) had some activity withTag-6P, it is probable that the activity in theLac- Gald strains was due to the Fru-6P kinaseof the glycolytic pathway. This is supported bykinetic studies (this study) which showed thatthe ketohexose 6-phosphate kinase in crude cellextracts of C10 Lac- Gald had similar propertiesto the purified Fru-6P kinase from Cl0. It istherefore likely that the lactose plasmid codesfor all three enzymes of the Tag-6P pathway.However, other possibilities cannot be ruledout. For example, the lactose plasmid may codefor a regulatory gene(s) that is essential for thechromosomal expression of the three enzymes.The lactose plasmid may code for different

genes in different strains. In S. lactis C2F (26)and S. cremoris B1 (1), the phospho-,B-galacto-sidase-and the ,B-galactoside-specific EII andFIII proteins appear to be plasmid associated.Work with S. lactis ATCC 11454 (strainDR1251) (20, 21) supports this, though the na-ture of the transport defect in Lac- Gald strainswas not investigated. More conclusive proofwas provided by enzymatic and molecular anal-ysis of the transformants, as genes for the lac-tose-PTS and phospho-,-galactosidase weretransferred on an S. lactis plasmid to a doubleLac- mutant of Streptococcus sanguis Challis(32). In the three strains that we studied, inaddition to deletion of the Tag-6P enzymes, the

activities for both the lactose-PTS and phospho-P-galactosidase were undetectable or deficientin the Lac- Gald derivatives, consistent with aplasmid association of these two activities. Thegalactose-PTS was detectable in the Lac- Galdderivatives, and with strain 133, the Km forgalactose was independent of the Lac pheno-type. Thus, the galactose-PTS is distinct fromthe lactose-PTS, as indicated by other workers(28, 38). Our evidence does not rule out thepossibility that the lactose-PTS has some activi-ty with galactose. If this is the case, then instrain 133 the kinetics with galactose substratemust be very similar for both the galactose-PTSand the lactose-PTS. Lactose-PTS activity withgalactose could explain the lower specific activi-ties of the galactose-PTS in the Lac- Galdderivatives. On the other hand, the lactose-PTSmay have no activity with galactose, and thelower specific activities may result from anabsence of the three Tag-6P pathway enzymes.In the Lac- Gald derivative of C1o, lactose-PTSactivity and phospho-,-galactosidase activitywere detectable, although specific activitieswere lower than in the wild type. Gene duplica-tion could explain these findings. There is evi-dence for a second phospho-p-galactosidasegene association with the chromosome in S.cremoris (1).

In S. lactis 133 and H1 (and probably C10),there appear to be six proteins associated withthe lactose plasmid. The six proteins are Gal-6Pisomerase, Tag-6P kinase, TDP aldolase, phos-pho-,B-galactosidase, and the lactose-specificPTS proteins FIll and EII (Fig. 2). We haveassumed that the deficient lactose-PTS in strains133 and H1 (and probably Cl0) is due to theabsence of the lactose-specific ElI and FIIIproteins, as has been shown in other lacticstreptococci (1, 26). The minimum size of DNArequired to code for the proposed six proteinswas calculated as approximately 6 to 7 Mdal,whereas the lactose plasmid size was between 30and 40 Mdal in most of the lactic streptococcalstrains studied (1, 17, 18, 21, 31; this study). Thecalculation was based on data for the relevantproteins from either lactic streptococci (8, 16;A. M. Fordyce, Ph.D. thesis) or S. aureus (6,29, 30) (enzymes common to both S. aureus [5,13] and lactic streptococci [8, 16] are similar insize).

S. lactis CI0 and H1 (this study) and indepen-dently derived Lac- Gald strains of S. lactis 133(ATCC 11454) (11, 21; this study) grew ongalactose with longer generation times and shift-ed to a more heterolactic fermentation comparedwith their Lac' parents. Our work provides apossible explanation for how the Lac- Galdphenotype influences the metabolism of galac-tose. In the three strains studied, the lactose

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PTS3

WE It, F XI

i..ac.t;nsi,- Fl

Phospho- 3 -galactosidase

+ ;e .r" 1roxgaCEIflIwN-- ,

!DP

TDP aldolase

'- i Ey t8tI(T F dile-3-!

FIG. 2. Enzymes (in italics) considered to be associated with the lactose plasmid in S. lactis. Gal-6P (*)

accumulated to high concentrations in Lac GaP' strains growing on galactose.

phenotype appears to be associated with a plas-mid that codes for the three Tag-6P pathwayenzymes. The galactose-grown Lac- Gald deriv-atives had no detectable activities of two, orpossibly three, of these enzymes, but still hadgalactose-PTS activity, though at a lower levelthan for Lac' cells grown on galactose. Theintracellular accumulation of Gal-6P to high con-centrations (up to 49 mM) in these mutants waspresumably due to the absence of Gal-6P isom-erase. Similar metabolite accumulation is foundin mutants of S. aureus (4). In the Lac' strains,the presence of intermediates and enzymes ofthe Tag-6P and Leloir pathways (Fig. 2) suggeststhat both pathways are actually operating incells growing on galactose, although their rela-tive contribution may depend upon the galactoseconcentration in the medium (36). In the Lac-Gald strains, galactose can only be metabolizedby the Leloir pathway. The slower growth rateof Lac- Gald mutants on galactose could be dueto accumulation of Gal-6P or growth-limitingLeloir pathway activity, or both. In contrast tothe present results, Gal-6P was not accumulatedin a Lac- Gald variant of S. lactis C2 (28). Themetabolic defect responsible for slow growth ongalactose (Gald) is thus strain dependent.

This study has shown that the metabolic alter-ations in some Lac- Gald strains of S. lactis aremore extensive than previously envisaged andhas provided some understanding of how theLac- phenotype influences galactose metabo-lism. The Lac- Gald phenotype in the three

strains studied was associated with either theabsence or a deficiency of the three Tag-6Ppathway enzymes, the lactose-PTS, and phos-pho-3-galactosidase. Lac- Gald cells still re-tained the ability to form Gal-6P via a deficientgalactose-PTS but could not further metabolizethis intermediate via the Tag-6P pathway. Thesecells therefore can only metabolize galactose bythe Leloir pathway, and the high intracellularconcentration of Gal-6P that accumulated mayreduce the doubling time. This could result in alower intracellular concentration of FDP, thuscausing the shift to a more heterolactic fermen-tation.

ACKNOWLEDGMENTS

We thank Judith Cleland and Gill Davies for excellenttechnical assistance and Alison Fordyce for helpfil discus-sion.

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TAG-6PF:AT1WrAA -W .-*/hvAY

(aiactose

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Gal-6P Isomerase

ag-6P kinase

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PLASMID-LINKED LACTOSE METABOLISM IN S. LACTIS 83

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