Vol. 142, No. 3JOURNAL OF BACTERIOLOGY, June 1980, p. 777-7850021-9193/80/06-0777/09$02.00/0

Physiological Studies of ,B-Galactosidase Induction inKluyveromyces lactis

ROBERT C. DICKSON* AND JENNIFER S. MARKINDepartment of Biochemistry, College ofMedicine, University of Kentucky, Lexington, Kentucky 40536

We examined the kinetics of /?-galactosidase (EC 3.2.1.23) induction in theyeast Kluyveromyces lactis. Enzyme activity began to increase 10 to 15 min,about 1/10 of a cell generation, after the addition of inducer and continued toincrease linearly for from 7 to 9 cell generations before reaching a maximum,some 125- to 150-fold above the basal level of uninduced cells. Thereafter, as longas logarithmic growth was maintained, enzyme levels remained high, but enzymelevels dropped to a value only 5- to 10-fold above the basal level if cells enteredstationary phase. Enzyme induction required the constant presence of inducer,since removal of inducer caused a reduction in enzyme level. Three nongratuitousinducers of fi-galactosidase activity, lactose, galactose, and lactobionic acid, wereidentified. Several inducers of the lac operon of Escherichia coli, includingmethyl-, isopropyl- and phenyl-1-thio-fl-D-galactoside, and thioallolactose did notinduce ,B-galactosidase in K. lactis even though they entered the cell. Themaximum rate ofenzyme induction was only achieved with lactose concentrationsof greater than 1 to 2 mM. The initial differential rate of fl-galactosidase appear-ance after induction was reduced in medium containing glucose, indicating tran-sient carbon catabolite repression. However, glucose did not exclude lactose fromK. lactis, it did not cause permanent carbon catabolite repression of fl-galactosid-ase synthesis, and it did not prevent lactose utilization. These three results are indirect contrast to those observed for lactose utilization in E. coli. Furthermore,these results, along with our observation that K. lactis grew slightly faster onlactose than on glucose, indicate that this organism has evolved an efficientsystem for utilizing lactose.

Kluyveromyces lactis (Saccharomyces [24]) isa budding yeast that can use lactose as a carbonand energy source. Lactose is hydrolyzed to glu-cose and galactose by an intracellular /8-galac-tosidase (EC 3.2.1.23) (6). This organism is in-teresting because 8i-galactosidase is induced bylactose and galactose (23), a phenomenon whichsuggests that these carbohydrates may be regu-lating expression of the f8-galactosidase struc-tural gene. Consequently, we chose to study themechanism by which lactose induces /-galacto-sidase in K. lactis because of the opportunity toexamine the molecular details of gene regulationin a eucaryote. Although this regulatory systemmight be substantially different from the well-characterized lactose (lac) operon of Esche-richia coli, it enables us to utilize some of thetechniques developed for studying the lac op-eron, such as chromogenic substrates for enzymeassays and a variety of substrate analogs forselection of mutants (4). Moreover, we can com-pare directly the genetic regulatory mechanismof a procaryote and a eucaroyte.Herman and Halvorson (8) examined the ge-

netics of fi-galactosidase synthesis in K. lactis.

I In a cross of two wild-type strains (a Y1140 xa Y1118), they found segregation ratios for ,B-galactosidase activity/nonactivity which sug-gested that there are two polymeric genes forfl-galactosidase, with strain Y1118 being LACIlac2 and strain Y1140 being lacl LAC2. Theyalso (8) demonstrated linkage between LACIand the structural gene for ,B-glucosidase instrain Y1118, whereas in strain Y1140 no linkagewas detected between LAC2 and the fl-glucosi-dase gene. Other studies have shown that ,B-galactosidase is induced four- to sixfold in sta-tionary-phase cells by lactose, galactose, andmethyl-l-thio-/8-D-galactoside (23). Finally, f8-galactosidase has been used as an enzymemarker for studies of the cell division cycle.Under noninducing conditions, the basal level offB-galactosidase doubles only during late G2phase of the cell cycle (20, 27). However, ifinducer is added at any phase of the cell cycle,enzyme activity is induced immediately (20).As part of our effort to determine the molec-

ular mechanism of/8-galactosidase induction, weexamined in detail the kinetics of enzyme ap-pearance after induction, the types of com-

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778 DICKSON AND MARKIN

pounds that induce, and other factors, such asglucose, which might affect the induction mech-anism. The results of these studies are presentedhere.

MATERIALS AND METHODSCell strains and culture media. K. lactis NRRL

a Y1140 (8) was used for all experiments. Cells weremaintained on slants of YM medium supplementedwith 10 g of glucose and 30 mg of adenine sulfate perliter. Complex YM medium (25) contained the follow-ing, per liter: 3 g of malt extract, 5 g of peptone, and 3g of yeast extract. Yeast nitrogen base medium withamino acids (Difco Laboratories) was prepared accord-ing to the manufacturer's directions, except that theconcentration was doubled (2x YNB). Another de-fined medium (DS medium) contained the following,per 100 ml: 2 g of NH4H2PO4, 0.167 g of KH2PO4, 0.167g of NaH2PO4, 10,g each of adenine and uracil, 10 mgof CaCl2, 60 mg of MgSO4, 2 mg each of histidine,lysine, and tyrosine, 2 ,ug of biotin, 400 pg of calciumpantothenate, 2 Ag of folic acid, 2 ,ug of inositol, 400,ug of niacin, 200 ,ug ofp-aminobenzoic acid, 400 jig ofpyridoxal hydrochloride, 200 ,ug of riboflavin, 400 jg ofthiamine hydrochloride, and 10,ug each of boric acid,copper sulfate, potassium iodide, ferric chloride, so-dium molybdate, and zinc sulfate. The pH was ad-justed to 5.5 with NaOH. All media were supple-mented with a carbon source as indicated below.

Buffers and other reagents. Z buffer (16) con-tained 0.1 M sodium phosphate, 10 mM KCI, 1 mMMgSO4, and 50 mM 2-mercaptoethanol, pH 7.0. Z-1buffer had the same composition as Z buffer exceptthat the pH was adjusted to 8.2 with NaOH. Allchemicals were reagent grade unless noted otherwise.Methyl-, isopropyl-, and phenyl-l-thio-fl-D-galacto-side, ,-D-galactopyranosyl (1 -. 4)-D-gluconic acid(lactobionic acid), and o-nitrophenyl-,f-D-galactopy-ranoside were from Sigma Chemical Co. S-/-D-galac-topyranosyl (1 -. 6)-D-glucose (thioallolactose) was agift from M. Barkely and was originally obtained fromW. Boos.Enzyme assays. /8-Galactosidase was assayed

spectrophotometrically, with o-nitrophenyl-fl-D-galac-topyranoside as substrate, by the procedure of Miller(16) as modified below. Two procedures were used toassay the enzyme content of intact cells. In procedureA, samples of 100 pl or less were combined with 0.9 mlof Z buffer and enough culture medium to give a totalvolume of 1.0 ml. This procedure could not be usedfor larger volumes because the pH of the mixturedropped below 6.9. This problem was particularlyacute when samples of different sizes were assayed, asduring an induction experiment. To avoid this prob-lem, we developed procedure B, in which the assaymixture contained 0.5 ml of Z-1 buffer, the sample,and enough culture medium to give a final volume of1.0 ml.

Since K. lactis is only slightly permeable to o-nitro-phenyl-f-D-galactopyranoside, cells were madepermeable by blending the assay mixture in a Vortexmixer for 15 s with 3 drops of toluene and then shakingit for 60 to 90 min at 30°C on a gyratory shaker.Thereafter, assay mixtures were equilibrated for 5 min

J. BACTERIOL.

at 30°C, and the reaction was started by adding 0.2 mlof o-nitrophenyl-18-D-galactopyranoside (4 mg/ml inwater). Reactions were stopped by adding 0.5 ml of 1M sodium carbonate. Cells were removed by centrif-ugation, and the absorbance of the supernatant solu-tion at 420 nm (A420) was measured. One unit ofenzyme activity equals the production of 1 nmol of o-nitrophenol per min per ml. The molar extinctioncoefficient of o-nitrophenol under these conditions is4.5 x 103 M-' cm-' (16).

During an induction experiment, where sampleswere taken over an extended period of time, it wasconvenient to store and assay them in batches. Thiswas accomplished by immediately mixing the samplein a Vortex mixer for 15 s with toluene and Z-1 buffercontaining 0.04% sodium azide and chilled to 4°C.These samples could be stored at 4°C for up to 5 hwithout loss of enzyme activity. Thereafter, sampleswere shaken at 30°C and assayed as described above.Standard growth procedure. Cells in stationary

phase were obtained by growing cells from a slant in10 ml of medium for 16 to 20 h at 28, 30, or 36°C withcontinuous aeration. Log-phase cells were obtained bydiluting stationary-phase cells into fresh medium togive an Awo of 0.15 and growing for 1 cell doubling.

Miscellaneous procedures. Cell densities weremeasured spectrophotometrically at 600 nm with aZeiss PM Q2 spectrophotometer. For such measure-ments, cells were diluted to an A600 of less than 0.7;higher densities did not obey the Beer-Lambert func-tion. The number of cells per milliliter was determinedby counting cells in a Petroff-Hausser counting cham-ber. Under our experimental conditions in DS me-dium, there were 3.8 x 107 log-phase cells per A600unit. Control experiments showed that both cells permilliliter and DNA per milliliter were linear with Awomeasurements, indicating that the latter measurementwas an accurate measure of cell concentration. DNAwas measured with diphenylamine (22).

Incorporation of D-glucose-['4C]lactose (AmershamCorp.; CFA 278, 58 mCi/mmol) or [U-'4C]glncose(New England Nuclear Corp.; NEC-042B, 1 to 5mCi/mmol) into acid-insoluble material was measured bydiluting 0.5 ml of cells into 0.5 ml of carrier cells havingan A600 of 2. Cold 12.5% trichloroacetic acid (4 ml)containing 20 mM lactose and glucose was addedimmediately, and the sample was stored overnight at4°C. The sample was centrifuged for 3 min at 4,000 xg, and the cell pellet was suspended in 4 ml of cold10% trichloroacetic acid containing lactose and glu-cose. Centrifugation and suspension of cells were re-peated for a total of four times with no incubationperiod. Cells were then washed once in 10 ml of 95%ethanol before being suspended in 10 ml of toluene-base scintillation fluid containing 25% (vol/vol) TritonX-100 and 6.5% terphenyl. Samples were counted in ascintillation counter (Packard Instrument Co., Inc.;no. 3003).

RESULTSGrowth rate of K. Iactis. The growth rate

of K. lactis Y1140 on various media and carbonsources was measured to determine doublingtimes and the density at which logarithmic

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INDUCIBLE fl-GALACTOSIDASE IN YEAST 779

growth stops. This information was essential forthe design of experiments presented in the fol-lowing sections. Representative growth curvesare shown in Fig. 1, and a summary of data isgiven in Table 1. K. lactis grew fastest on lactose,with a doubling time of 91 min in DS mediumand 87 min in YM medium. Of the carbonsources tested, cells grew slowest on glycerol,and they only grew well on this compound attemperatures below about 30°C. In contrast, K.lactis grew on the other carbon sources listed inTable 1 at temperature as high as 370C; highertemperatures were not examined. This strain ofK. lactis could also use fructose, D-mannitol,lactobionic acid, L-sorbose, D-sorbitol, pyruvate,fumarate, succinate (8), and methyl-, isopro-pyl-, or phenyl-f8-D-galactoside as a carbonsource (data not shown). In DS medium, cit-rate, D- or L-fucose, L-arabinose, ethanol, andmethyl-, isopropyl-, or phenyl-l-thio-,8-D-galac-toside did not serve as carbon sources (data notshown).Rate of fi-galactosidase induction as a

function of inducer concentration. We de-termined the rate of increase in,f-galactosidaseactivity, hereafter referred to as the rate of /8-galactosidase induction, as a function of inducerconcentration to establish the concentration ofinducer which gives the maximal rate of induc-tion. Cells were grown in glycerol to avoid theproblem of transient carbon catabolite repres-sion (see below). Lactose was used as inducer in

1 2 3 4 5 6 7 8 9 10 11 12 13 14HOURS

FIG. 1. Growth curve of K. lactis strain Y1140.Stationary-phase cells were diluted into fresh DSmedium containing either 20 mM lactose (0) or sor-

bitol (V) as a carbon source and grown at 30°C on a

gyratory shaker. Cell density was measured spectro-photometrically at 600 nm.

TABLE 1. Growth rate ofK. lactis Y1140

Medium Carbon source' Doublingtime" (min)

DS Glycerol 165DS Glucose 106DS Sorbitol 144DS Lactose 91YM Glucose 100YM Lactose 872x YNB Glucose 1232x YNB Lactose 105

a The final concentration was 20 mM.b These values represent the average of two or more

experiments.

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FIG. 2. Rate of /3-galactosidase induction as afunction of inducer concentration. Cells were grownto stationary phase in DS medium containing 20mMglycerol at 28°C, diluted into fresh medium to give aninitial Ae,wo of 0.15, grown for 1 cell doubling, andthen induced at time zero with lactose. Final concen-trations (millimolar) of lactose were as follows: 0, 8;A, 2; [, 0.5; and V, 0.125.

all experiments. As shown in Fig. 2, the rate offl-galactosidase induction was maximal with lac-tose concentrations of greater than 2 mM. Slightinduction was observed during the 2-h assayperiod with 0.125 mM lactose. In another exper-iment, 1 mM lactose produced the maximal rateof enzyme induction. The data in Fig. 2 werealso plotted as differential rates of enzyme in-duction (units of enzyme per milliliter plottedagainst culture density, A6wo) (17) to obviate anydifferences in growth rates which might existamong the cultures due to the utilization oflactose (data not shown). The differential rate

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of enzyme induction was maximal with lactoseconcentrations above 1 mM. With 0.5 and 0.125mM lactose, the differential rate of inductionwas reduced to 1/2 and l/30, respectively. We con-clude from these experiments that the maximalrate of fi-galactosidase induction is achievedwith concentrations of lactose of greater than 1to 2 mM.

Effect of glucose on 8i-galactosidase in-duction. In E. coli, the synthesis of f3-galacto-sidase can be influenced by glucose in three ways(14). First, glucose can prevent the entry oflactose into the cell and therefore prevent in-duction, an effect termed inducer exclusion. Sec-ond, the presence of glucose in induced cellsreduces the differential rate of fl-galactosidasesynthesis by half, a phenomenon termed carboncatabolite repression (permanent repression).Third, the initial rate of,-galactosidase synthe-sis in induced cells is reduced by glucose, aphenomenon termed transient repression. Thelatter two effects of glucose on ,B-galactosidaseinduction result from positive regulation of thelac operon by the catabolite gene activator pro-tein and cyclic AMP (reviewed in reference 3).Carbon catabolite repression has also been ob-served in yeast (see below).We examined f?-galactosidase induction in K.

lactis to determine whether it was regulated bya glucose-sensitive system. Initially, we foundthat glucose did not exclude the entry of lactose,since cells grown in glucose could be induced bylactose (see below, Fig. 4b). The two other pos-sible effects of glucose, permanent and transientcarbon catabolite repression, were examined bygrowing cells in DS medium containing eitherglycerol or glucose as the carbon source andinducing ,l-galactosidase with lactose. As shownin Fig. 3, cells grown in glycerol and induced bylactose began to synthesize fl-galactosidase atthe maximal differential rate, whereas cellsgrown in glucose and induced by lactose did notbegin immediately to synthesize ,8-galactosidaseat the maximal differential rate. Instead, therewas a short lag period during which the differ-ential rate of enzyme synthesis increased untilthe maximal rate was reached. Thus, ,B-galacto-sidase synthesis in K. lactis showed transient,but not permanent, carbon catabolite repression.In this experiment, the differential rate of en-zyme synthesis for induced glucose-grown cellswas 43 times greater than that for uninducedcells. This value varied from 33 to 100, with anaverage of 58. Variability was mainly due toinaccuracy in measuring the low enzyme levelsin uninduced cells. Cells grown on sorbitolshowed almost no transient repression (data notshown).

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othiaIUo0I.-I

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A600nmFIG. 3. Transient repression of,#-galactosidase in-

duction. The effect ofglucose on the rate of /3-galac-tosidase induction was examined by comparing thedifferential rate of enzyme induction between cellsgrown on glucose and those grown on glycerol. Cellswere grown to stationary phase in DS medium con-taining 20 mM glycerol or glucose at 28°C, dilutedinto fresh medium, allowed to double, and inducedwith 20mM lactose at an Aw0o of 0.32 ± 0.02. Cultureswere as follows: 0, glycerol-grown plus lactose; A,glucose-grown plus lactose; O, glycerol- or glucose-grown without lactose.

Effect of glucose on utilization of lactose.The foregoing experiments demonstrate thatglucose does not block the induction of 8-galac-tosidase. The experiments do not, however, in-dicate whether lactose is used as a carbon sourceafter enzyme induction. For example, it is con-ceivable that lactose transport might be limitedby the presence of glucose and that cells wouldbe unable to use lactose even though ,8-galacto-sidase is abundant. The data in Fig. 4a (cultureC) showed that cells growing in the presence of20 mM glucose and induced at time zero by theaddition of 20 mM ['4C]lactose began to incor-porate 14C into acid-insoluble material within 60min. A control experiment demonstrated thatincorporation of ['4C]lactose required inductionsince there was no incorporation in an uninduced

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FIG. 4. Kinetics oflactose utilization in cellsgrow-ing on glucose. Stationary-phase cells were grown on

the carbon source indicated below in DS medium anddiluted into 8.5 ml of fresh medium to an A., of 0.2.After 2 h of growth at 28°C, the experiment was be-gun (time zero) by adding 1.5 ,Ci of ['4Cilactose or

['4Ciglucose along with a nonradioactive carbohy-drate as indicated below. Incorporation of radioac-tivity into acid-soluble material (a) was measured as

described in the text. (3-Galactosidase activity (b) wasmeasured by procedure A. Cultures were as follows:A (0), grown on 20 mM glucose, ['4Ciglucose added;B (5), grown on 20mM glucose, ['4C]glucose and 20mM lactose added; C (V), grown on 20 mM glucose,['4Cilactose and 20mM lactose added; D (A), grownon 20mM lactose, [14Cflactose added; E (*), grownon 20mM glucose, ['4Cilactose added (final concen-

tration of lactose, 6 uM).

glucose-grown culture containing 6 ,uM [4C]ilac-tose (culture E, Fig. 4a). This low concentrationoflactose did not induce ,/-galactosidase activity,whereas 20 mM lactose did (Fig. 4b). We con-

clude that glucose does not prevent either theuptake or the utilization of lactose.We further examined the utilization of lactose

by comparing the rate of ['4C]glucose accumu-lation in uninduced and induced glucose-growncultures. If lactose is utilized by the inducedculture, there should be less accumulation of["4C]glucose. The data shown in Fig. 4a, culturesA and B, verified this prediction and supportedour conclusion that lactose can be utilized in thepresence of glucose. To determine whether glu-cose or lactose is preferentially utilized, theamount of glucose incorporated per A6w unitwas measured after long-term labeling. After 6and 12 cell doublings, a glucose-grown (20 mM[I4C]glucose) culture incorporated twice as manymillimoles of glucose as did a glucose-grownculture containing 20 mM lactose. We concludethat there was no preferential utilization of glu-cose or lactose in a culture containing both car-bohydrates.Kinetics of P-galactosidase induction. In-

ducible and derepressible enzyme activities inyeast generally increase within a few minutesafter induction or derepression. However, this isnot true for some enzymes, particularly thoseinvolved in long-term adaptation (21, 26) inwhich several cell generations pass before en-zyme activity begins to increase.The kinetics of f8-galactosidase appearance

after induction with lactose were examined in K.lactis to determine whether enzyme activity ap-pears immediately after the addition of induceror whether there is a long lag. For these experi-ments, cells were grown to early log phase in DSmedium containing sorbitol and then inducedwith lactose. Sorbitol was used as a carbonsource because it gave the lowest basal level of,B-galactosidase (see below, Table 2); cells grewrapidly, and only a slight transient repressionwas observed. As the insert in Fig. 5 shows, ,B-galactosidase activity started to increase 10 to15 min after the addition of inducer at 300C.Enzyme activity continued to increase for about7 h (3 to 4 cell doublings) or until a cell densityof 5 to 7 A6w units per ml was reached; thereaf-ter, enzyme activity decreased (Fig. 5). Althoughsix induction experiments of this type gave sim-ilar results, there was variability in the shape ofthe curve. For example, peak enzyme activitiesranged from 50- to 100-fold above the level in anuninduced culture; the peak occurred at a celldensity of as low as 4 and as high as 9.5 A6wunits per ml, and the decrease in enzyme activityoccurred at a lower rate. Variation in the induc-tion ratio was due to a twofold day-to-day dif-ference in basal enzyme levels. The reasons forthe other variations are unknown.

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FIG. 5. Kinetics of ,B-galactosidase induction.Standard log-phase cells were induced with 20 mMlactose at time zero. Thereafter, cell density andenzyme activity were measured by procedure B asdescribed in the text. In one experiment, cells wereallowed to grow to stationary phase (A). In anotherexperiment, cells were kept in log phase by repeateddilution with fresh medium (0). Early timepoints areshown in the insert. Experimental details are givenin the text.

In the preceding experiment, it appeared asthough enzyme activity began to decrease ascells approached stationary phase. Conse-quently, the maximum or plateau enzyme con-tent per cell may not have been reached. Weexamined this possibility with cells kept in logphase at an A6co of between 0.3 and 0.7 byrepeated dilution with fresh medium. Underthese conditions, enzyme activity increased forabout 12 h (7 to 9 cell doublings) before reachinga maximum value approximately 100-fold higherthan the uninduced level (Fig. 5). Again, sixexperiments of this type gave similar results, butthere was variability. For instance, maximumenzyme activity was reached in as short a timeas 11 h and as long as 14 h, maximum enzymelevels ranged from a low of 49 to a high of 132above the uninduced level, and maximum en-zyme levels remained constant rather than de-creasing 30% as shown in Fig. 5. The reasons forthese differences remain unknown.

It is clear from the preceding experiments thatmaximal ,f-galactosidase levels per cell are onlyreached if cells are held in log phase for 11 to 14

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h in the presence of inducer (7 to 9 cell dou-blings); in cells reaching stationary phase beforethat number of cell doublings, enzyme activitystops short of the maximum level and begins todecrease rapidly.Inducer withdrawal. In the preceding ex-

periments, ,B-galactosidase induction was meas-ured on cells exposed continuously to inducer.In these experiments, we examined the responseof enzyme levels in induced cells to the with-drawal of inducer. As shown in Fig. 6, f,-galac-tosidase levels stopped increasing and starteddecreasing within 10 min after inducer was with-drawn. Enzyme levels may start decreasing inless than 10 min, but this would not be deter-mined since 5 to 7 min elapsed between inducerremoval and suspension of cells in fresh medium.We concluded from this experiment that enzymelevels increase only in the presence of inducer.Compounds that induce f8-galactosidase.

We examined the types of carbohydrates thatinduce f8-galactosidase to characterize furtherthe induction process. Initially, enzyme levelswere measured on cells grown for 18 h at 300Cto stationary phase in the presence or absenceof inducer. This approach proved unacceptablebecause: (i) ideally, we wanted to measure max-imum induction levels, but as can be seen fromthe induction curve shown in Fig. 5, measure-

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FIG. 6. Effect of inducer removal on /3-galactosid-ase induction. Log-phase cells growing at 30°C in DSmedium containing 20 mM glucose were induced for,3-galactosidase by adding lactose to 20 mM at -60min. At time zero, medium was removed by filtration(0.45-ttm membrane filter, type HA, Millipore Corp.),and cells were washed once with 0.5 volume of me-dium lacking lactose, suspended in fresh mediumcontaining glucose, and divided into two cultures.Lactose was added back to one culture (l), but not tothe other (0). /3-Galactosidase was assayed by pro-

cedure B as described in the text.

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INDUCIBLE f8-GALACTOSIDASE IN YEAST 783

ments made at 18 h did not represent maximumenzyme levels; and (ii) enzyme levels varied upto 10-fold from one experiment to the next forcells grown in the presence of compounds thatinduce, including lactose, galactose, and lacto-bionic acid. This variation was due in part todifferences in the rate at which enzyme levelsdecreased after reaching a maximum (Fig. 5).Because of these drawbacks, enzyme levels weremeasured on log-phase cells grown in the pres-ence of inducer from an A6eo of 0.5 to an A6eo of3 to 5 (about 3 cell doublings). Under theseconditions, /8-galactosidase levels approachedmaximum inducible levels as shown in Fig. 5.This procedure proved to be more acceptablebecause the variability of enzyme levels in in-duced cultures was reduced and the basal en-zyme level in uninduced cultures was reducedtwo- to fivefold, yielding higher induction valueswhen enzyme levels of induced and uninducedcultures were compared.Only three of the compounds tested, lactose,

galactose, and lactobionic acid, induced ,8-galac-tosidase (Table 2). All three compounds gavesimilar levels of induction, ranging from 116-foldfor lactobionic acid to 153-fold for galactose.Isopropyl-l-thio-f3-D-galactoside and thioallo-lactose showed twofold induction values, butthis could have been due to contaminating ga-lactose. For example, if 1% of either compoundhad been hydrolyzed to galactose, then the freegalactose concentration would have been lo-'M. As the control experiment in Table 2 shows,10-4 M galactose induced /1-galactosidase two-fold. We did not examine the purity of the car-bohydrates listed in Table 2; therefore, it ispossible that those showing low levels of induc-tion could have been contaminated by galactose.

Conceivably, thioallolactose and methyl-,isopropyl-, and phenyl-l-thio-,8-D-galactoside,which are good gratuitous inducers of the E. colilac operon (3, 11), failed to induce in K. lactisbecause of exclusion from the cell. This possibil-ity was examined by measuring the intracellularconcentration of radioactive methyl- and isopro-pyl-l-thio-fl-D-galactoside. Neither compoundwas excluded from K. lactis since both accu-mulated to as high an intracellular concentrationas lactose (data not shown). Similar experimentswith thioallolactose and phenyl-l-thio-ft-D-gal-actoside have not been done.Although galactose was a good inducer, its 6-

deoxy analog, D- and L-fucose, did not induce.K. lactis cannot use either of these compoundsas a carbon source; therefore, their intracellularaccumulation was not examined. The three com-pounds that induce, lactose, galactose, and lac-tobionic acid, can be used as carbon sources;

TABLE 2. Levels of /3-galactosidase elicited bycarbohydratesa

Carbohydrate

LactoseGalactoseLactobionic acidGalactose (10-4M)IPTGbMTGbfTGbThiallolactoseD-FucoseL-FucoseD-SorbitolGlucose

U of 8l-galacto-sidase per A6a

(avg)86.5 ± 6.9105 ± 5479.8 ± 171.32 ± 0.461.06 ± 0.340.67 ± 0.210.82 ± 0.201.22 ± 0.380.83 ± 0.230.63 ± 0.350.69 ± 0.210.90 ± 0.38

Ratio tosorbitol

126153116

1.91.50.971.21.81.20.911.01.3

a Standard stationary-phase cells were grown at30°C in DS medium containing 20 mM sorbitol and a20 mM concentration of the indicated carbohydrateunless noted otherwise. Cells were diluted into freshmedium and grown from 0.5 to 3 to 5 A6w units. Aftertreatment with toluene, f8-galactosidase was assayedby procedure A as described in the text. Averagevalues represent three or four experiments.

b IPTG, Isopropyl-l-thio-/i-D-galactoside; MTG,methyl-1-thio-fl-D-galactoside; 4TG, phenyl-l-thio-,B-D-galactoside.

therefore, no gratuitous inducer has yet beenfound.

DISCUSSIONOur examination of the kinetics of /8-galacto-

sidase induction demonstrated that enzyme ac-tivity began increasing 10 to 15 min after theaddition of inducer (lactose) at 30°C (Fig. 5).Thus, as with other inducible yeast enzymes (1,5, 8, 13), the induction of f8-galactosidase in K.lactis is a rapid process, requiring about l/Ao of acell division cycle or less. Induction of f,-galac-tosidase is unlike long-term enzyme adaptation(21, 26), in which many cell generations elapsebetween the addition of inducer and the appear-ance of enzyme. There are undoubtedly manyevents which occur during the 10- to 15-min lagafter the addition of inducer. Thus far, we haveidentified one of these events, which involveslactose uptake. Figure 4 shows that the uptakeof lactose requires induction. In other experi-ments, we have shown that the uptake system isindeed inducible by lactose; it requires energy,it is saturable, and it is blocked by genetic mu-tants, suggesting involvement of a specific per-mease (R. C. Dickson and K. Barr, unpublisheddata).

It is clear from our data (Fig. 5) that themaximum inducible /1-galactosidase level was

only achieved if cells were grown for 7 to 9 cell

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doublings in the presence of inducer. If cellsentered stationary phase before the necessarynumber of cell divisions, the maximum ,8-galac-tosidase level was not obtained, and the enzymelevel decreased. Likewise, the enzyme level de-creased shortly after inducer was removed fromthe culture (Fig. 6). The 80 to 90% decrease inenzyme level which occurred as cells approachedstationary phase (Fig. 5) could be explained bydilution due to cell division in the absence of netenzyme synthesis. Alternatively, the enzymelevel could decrease because of enzyme inacti-vation.Because glucose is a preferred carbon source

for numerous organisms, many inducible en-zymes cannot be induced when cells are grownin the presence of glucose. This phenomenon,termed carbon catabolite repression, has beenanalyzed most thoroughly for /3-galactosidaseinduction in E. coli (reviewed in reference 14).Similar effects of glucose on inducible enzymeshave been noted in yeast. For example, Adams(1) demonstrated that galactokinase inductionin Saccharomyces cerevisiae was sensitive toboth permanent and transient carbon cataboliterepression. Recently, Matern and Holzer (15)showed that the addition of glucose to S. cere-

visiae grown on galactose results in an inacti-vation of the galactose uptake system. Otherglucose-sensitive inducible enzymes in S. cere-

visiae include maltase and other a-glucosidases(28), invertase (29), alcohol dehydrogenase (5),catalase (19), and enzymes of the citric acid cycle(18).Our results demonstrated that glucose caused

a slight transient repression of the initial differ-ential rate of fi-galactosidase synthesis (Fig. 3),but it had no effect on the final differential rateof synthesis, nor did it prevent lactose uptake or

utilization (Fig. 4). Thus, there is no permanentcarbon catabolite repression of ,B-galactosidasesynthesis or lactose utilization in K. lactis. In-duction of /8-galactosidase activity and lactoseutilization in K. lactis and E. coli clearly responddifferently to the presence of glucose. The lackof permanent carbon catabolite repression andinducer exclusion can be explained in two ways;

neither explanation excludes the other, and theymay both apply. The first explanation is teleo-logical and argues that not only glucose but alsolactose is a preferred carbon source becausedairy products are the natural habitat for K.lactis (24). This possibility was supported byour data showing that K. lactis grew faster on

lactose than on glucose (Table 1). The fastergrowth rate on lactose could have been due tofaster uptake or to the availability of galactose;either or both possibilities could limit the growth

rate on glucose. The second explanation for theabsence of permanent carbon catabolite repres-sion and inducer exclusion argues that K. lactislacks such a repression mechanism. In this re-gard, only one other K. lactis enzyme, f?-glucos-idase, has been examined for carbon cataboliterepression; it is atypical because low concentra-tions (less than 1 mM) of glucose induce,whereas higher concentrations (greater than 10mM) repress enzyme levels by 40% (9). Since K.lactis can use galactose as a carbon source, itwould be informative to determine whether thegalactose catabolic enzymes are repressible byglucose as they are in S. cerevisiae (1, 15).We examined the types of compounds that

induce /3-galactosidase to begin characterizingthe chemical nature of the natural inducer, tofind a gratuitous inducer, and to compare thisregulatory system with the lac operon of E. coli.Two inducers of,-galactosidase in K. lactis,lactose and galactose, are also inducers of thelac operon (3, 11), whereas lactobionic acid in-duces K. lactis but not the lac operon (12).Surprisingly, several good inducers of the lacoperon (3, 11), including isopropyl-, methyl-, andphenyl-l-thio-,8-D-galactoside and thioallolac-tose, did not induce K. lactis. Since isopropyl-and methyl-l-thio-,8-D-galactoside accumulatein K. lactis cells to as high a concentration asthat of lactose, their failure to induce is not dueto exclusion from the cell, but could be due tointracellular inactivation. In E. coli, isopropyl-1-thio-,8-D-galactoside is ultimately inactivated byacetylation (2). We did not examine this possi-bility directly. However, since K. lactis cannotuse either of these compounds as a carbonsource, it seems unlikely that they are metabo-lized to any large extent, but minor chemicalmodifications need to be analyzed. The oxy an-alog of these two compounds, methyl- and iso-propyl-f?-D-galactoside, induce /8-galactosidase,but this may be due to a release of galactosesince K. lactis can use either compound as acarbon source. Although these data demonstratea clear difference between the responsiveness ofK. lactis and E. coli to inducers, it is still possiblethat the natural inducer of the lac operon, allo-lactose, is also the natural inducer in K. lactis.We have shown that purified K. lactis fl-galac-tosidase does make allolactose during hydrolysisof lactose (6), as has been noted with E. coli f3-galactosidase (10). Positive identification of thenatural inducer will require experiments withmutants and purified cellular components, ashas been done for the lac operon (11).

Tingle and Halvorson (23) measured ,B-galac-tosidase induction in strain Y1118 of K. lactis, astrain related to the strain that we used. They

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found that for stationary cells grown in thepresence of 6mM galactose, 3 mM lactose, or 10mM methyl-l-thio-,B-D-galactoside, ,B-galacto-sidase was induced 6.4-, 4.6-, and 5-fold, respec-tively. Their induction values for galactose andlactose are lower than our values (49- and 23-fold, respectively [data not shown]), whereastheir value for methyl-l-thio-,8-D-galactoside ishigher than our value of 2 (Table 2). Thesedifferences may be due to strain differences, butthis remains to be determined.These results give an overview of the kinetics

of ,B-galactosidase induction and provide us witha framework for examining the mechanism ofinduction by biochemical and genetic ap-proaches (7).

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant

GM-22749 and Biomedical Research Support grant RR05374-15 from the National Institutes of Health. R.C.D. was sup-ported by Faculty Research Award no. FRA-183 from theAmerican Cancer Society.

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3. Barkley, M. D., and S. Bourgeois. 1977. The Lac op-eron, p. 66-127. In J. C. Copeland and G. A. Marzluf(ed.), Regulatory biology. Academic Press, Inc., NewYork.

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