transport and hydrolysis of disaccharides trichosporon ... · t. cutaneumtransport andhydrolysis of...

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Vol. 168, No. 2 Transport and Hydrolysis of Disaccharides by Trichosporon cutaneum MONIKA MORTBERG AND HALINA Y. NEUJAHR* Department of Biochemistry and Biotechnology, The Royal Institute of Technology, S-100 44 Stockholm, Sweden Received 13 January 1986/Accepted 28 July 1986 Trichosporon cutaneum is shown to utilize six disaccharides, cellobiose, maltose, lactose, sucrose, melibiose, and trehalose. T. cutaneum can thus be counted with the rather restricted group of yeasts (11 to 12% of all investigated) which can utilize lactose and melibiose. The half-saturation constants for uptake were 10 3 mM sucrose or lactose and 5 1 mM maltose, which is of the same order of magnitude as those reported for Saccharomyces cerevisiae. Our results indicate that maltose shares a common transport system with sucrose and that there may be some interaction between the uptake systems for lactose, cellobiose, and glucose. Lactose, cellobiose, and melibiose are hydrolyzed by cell wall-bound glycosidase(s), suggesting hydrolysis before or in connection with uptake. In contrast, maltose, sucrose, and trehalose seem to be taken up as such. The uptake of sucrose and lactose is dependent on a proton gradient across the cell membrane. In contrast, there were no indications of the involvement of gradients of H+, KV, or Na+ in the uptake of maltose. The uptake of lactose is to a large extent inducible, as is the corresponding glycosidase. Also the glycosidases for cellobiose, trehalose, and melibiose are inducible. In contrast, the uptake of sucrose and maltose and the corresponding glycosidases is constitutive. The uptake of disaccharides by yeast has been studied almost exclusively in strains of Saccharomyces cerevisiae (for review, see reference 2). Until quite recently, there was uncertainty whether disaccharides can be taken up as such or whether they must be hydrolyzed before uptake. How- ever, there is now unequivocal proof of the existence in S. cerevisiae of specific transport systems for sucrose (28) and maltose, the latter being inducible (29). The soil yeast Trichosporon cutaneum is important because of its extraor- dinary inducible capacity to metabolize phenols (10, 23, 25, 35). The flexibility of this organism with respect to various aromatic substrates and gratuitous synthesis of related as well as unrelated enzymes has been amply demonstrated (1, 11-13, 24, 26, 27, 30, 31, 34). In a previous report we demonstrated a specific, energy-dependent, and inducible transport system for phenol in T. cutaneum (21). In this paper we deal with the ability of T. cutaneum to utilize six different disaccharides as sole carbon sources. The results of studies on their transport, hydrolysis, and growth-promoting efficiency are presented. MATERIALS AND METHODS Chemicals and equipment. All chemicals were commercial products of reagent grade, and they were purchased as described previously (24). Triton X-100 was from Rohm & Haas (Philadelphia, Pa.), '4C-labeled compounds were from the Radiochemical Centre (Amersham, England), and Biofluor scintillation counting solution was from New En- gland Nuclear Corp. (Boston, Mass.). The inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP), gramicidin, and oligomycin were products of Boehringer GmbH (Mannheim, Federal Republic of Germany). Valinomycin, monensin, o-nitrophenyl-3-D-galactopyrano- side, methyl-ct-D-glucopyranoside (a-MG), and enzymes came from Sigma Chemical Co. (St. Louis, Mo.). NovoZym 234 was from NOVO Biolabs (Bagsvaerd, Denmark). Mem- * Corresponding author. brane filters were from Millipore Corp. (Moisheim, France). A Clark oxygen electrode (YSI 4004; Yellow Springs Instru- ment Co., Yellow Springs, Ohio) was used to measure oxygen consumption. Organism and culture conditions. T. cutaneum was grown at 28°C on a rotary shaker in the medium described by Neujahr and Varga (25) containing salts and 1 mg of thiamine hydrochloride per liter. The cells were grown for 16 to 18 h with 0.5% maltose, lactose, sucrose, cellobiose, melibiose, or trehalose. To obtain repressed cells, 2% glucose was added to the growth medium. The cells were collected by centrifugation at 5,000 x g at 4 to 8 °C and washed twice with distilled water. Uptake studies. All cell preparations were harvested dur- ing the exponential phase of growth and used for uptake studies on the same day. The uptake experiments were essentially as described previously (21). Cells were incu- bated in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid) (pH 7.5) with either uniformly 14C- labeled maltose or sucrose or D-glucose-[1-_4C]lactose and collected by filtration through Millipore membrane filters (0.45 ,um). The filters were then transferred to vials for liquid scintillation counting. The parameters of uptake were deter- mined by kinetic analysis with an HP-85 desk computer and the nonlinear regression algorithm of Marquardt (20, 22). Permeabilization. Cells were permeabilized by the method Miozzari et al. (20a), as modified by Mortberg and Neujahr (21). Preparation of protoplasts. Protoplasts were prepared as described by Stephen and Nasim (33) with about 2 h of incubation with NovoZym 234. The protoplasts were har- vested by gentle centrifugation and washed twice with 1.2 M sorbitol containing 20 mM MES [2-(N-morpholino) ethanesulfonic acid], pH 6.0. The supernatants from each step were collected for enzyme measurements. The protoplasts obtained after the second wash were lysed by osmotic shock in 0.1 M MES (pH 6.0). Enzyme assays. Glycosidase activities were determined as glucose liberated by intact cells, permeabilized cells, and 734 JOURNAL OF BACTERIOLOGY, Nov. 1986, P. 734-738 0021-9193/86/110734-05$02.00/0 Copyright X) 1986, American Society for Microbiology on August 10, 2019 by guest http://jb.asm.org/ Downloaded from

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Page 1: Transport and Hydrolysis of Disaccharides Trichosporon ... · T. CUTANEUMTRANSPORT ANDHYDROLYSIS OF DISACCHARIDES 735 LI-04 0 cr-5 10 15 > [LACTOSE] mM E 10 15 20 MALTOSE], mM 5 10

Vol. 168, No. 2

Transport and Hydrolysis of Disaccharides byTrichosporon cutaneum

MONIKA MORTBERG AND HALINA Y. NEUJAHR*

Department of Biochemistry and Biotechnology, The Royal Institute of Technology, S-100 44 Stockholm, Sweden

Received 13 January 1986/Accepted 28 July 1986

Trichosporon cutaneum is shown to utilize six disaccharides, cellobiose, maltose, lactose, sucrose, melibiose,and trehalose. T. cutaneum can thus be counted with the rather restricted group of yeasts (11 to 12% of allinvestigated) which can utilize lactose and melibiose. The half-saturation constants for uptake were 10 3 mMsucrose or lactose and 5 1 mM maltose, which is of the same order of magnitude as those reported forSaccharomyces cerevisiae. Our results indicate that maltose shares a common transport system with sucroseand that there may be some interaction between the uptake systems for lactose, cellobiose, and glucose.Lactose, cellobiose, and melibiose are hydrolyzed by cell wall-bound glycosidase(s), suggesting hydrolysisbefore or in connection with uptake. In contrast, maltose, sucrose, and trehalose seem to be taken up as such.The uptake of sucrose and lactose is dependent on a proton gradient across the cell membrane. In contrast,there were no indications of the involvement of gradients of H+, KV, or Na+ in the uptake of maltose. Theuptake of lactose is to a large extent inducible, as is the corresponding glycosidase. Also the glycosidases forcellobiose, trehalose, and melibiose are inducible. In contrast, the uptake of sucrose and maltose and thecorresponding glycosidases is constitutive.

The uptake of disaccharides by yeast has been studiedalmost exclusively in strains of Saccharomyces cerevisiae(for review, see reference 2). Until quite recently, there wasuncertainty whether disaccharides can be taken up as suchor whether they must be hydrolyzed before uptake. How-ever, there is now unequivocal proof of the existence in S.cerevisiae of specific transport systems for sucrose (28) andmaltose, the latter being inducible (29). The soil yeastTrichosporon cutaneum is important because of its extraor-dinary inducible capacity to metabolize phenols (10, 23, 25,35). The flexibility of this organism with respect to variousaromatic substrates and gratuitous synthesis of related as

well as unrelated enzymes has been amply demonstrated (1,11-13, 24, 26, 27, 30, 31, 34). In a previous report we

demonstrated a specific, energy-dependent, and inducibletransport system for phenol in T. cutaneum (21). In thispaper we deal with the ability of T. cutaneum to utilize sixdifferent disaccharides as sole carbon sources. The results ofstudies on their transport, hydrolysis, and growth-promotingefficiency are presented.

MATERIALS AND METHODS

Chemicals and equipment. All chemicals were commercialproducts of reagent grade, and they were purchased as

described previously (24). Triton X-100 was from Rohm &Haas (Philadelphia, Pa.), '4C-labeled compounds were fromthe Radiochemical Centre (Amersham, England), andBiofluor scintillation counting solution was from New En-gland Nuclear Corp. (Boston, Mass.). The inhibitorscarbonyl cyanide m-chlorophenylhydrazone (CCCP),gramicidin, and oligomycin were products of BoehringerGmbH (Mannheim, Federal Republic of Germany).Valinomycin, monensin, o-nitrophenyl-3-D-galactopyrano-side, methyl-ct-D-glucopyranoside (a-MG), and enzymes

came from Sigma Chemical Co. (St. Louis, Mo.). NovoZym234 was from NOVO Biolabs (Bagsvaerd, Denmark). Mem-

* Corresponding author.

brane filters were from Millipore Corp. (Moisheim, France).A Clark oxygen electrode (YSI 4004; Yellow Springs Instru-ment Co., Yellow Springs, Ohio) was used to measureoxygen consumption.Organism and culture conditions. T. cutaneum was grown

at 28°C on a rotary shaker in the medium described byNeujahr and Varga (25) containing salts and 1 mg of thiaminehydrochloride per liter. The cells were grown for 16 to 18 hwith 0.5% maltose, lactose, sucrose, cellobiose, melibiose,or trehalose. To obtain repressed cells, 2% glucose was

added to the growth medium. The cells were collected bycentrifugation at 5,000 x g at 4 to 8 °C and washed twice withdistilled water.Uptake studies. All cell preparations were harvested dur-

ing the exponential phase of growth and used for uptakestudies on the same day. The uptake experiments were

essentially as described previously (21). Cells were incu-bated in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.5) with either uniformly 14C-labeled maltose or sucrose or D-glucose-[1-_4C]lactose andcollected by filtration through Millipore membrane filters(0.45 ,um). The filters were then transferred to vials for liquidscintillation counting. The parameters of uptake were deter-mined by kinetic analysis with an HP-85 desk computer andthe nonlinear regression algorithm of Marquardt (20, 22).

Permeabilization. Cells were permeabilized by the methodMiozzari et al. (20a), as modified by Mortberg and Neujahr(21).

Preparation of protoplasts. Protoplasts were prepared as

described by Stephen and Nasim (33) with about 2 h ofincubation with NovoZym 234. The protoplasts were har-vested by gentle centrifugation and washed twice with 1.2 Msorbitol containing 20 mM MES [2-(N-morpholino)ethanesulfonic acid], pH 6.0. The supernatants from eachstep were collected for enzyme measurements. Theprotoplasts obtained after the second wash were lysed byosmotic shock in 0.1 M MES (pH 6.0).Enzyme assays. Glycosidase activities were determined as

glucose liberated by intact cells, permeabilized cells, and

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FIG. 1. Uptake of lactose, tnaltose, and sucrose in T. cutaneum.Uptake of '4C-labeled substrates was measured in 0.1 M HEPES(pH 7.5) after 1 min of incubation at 28°C. Main diagrams show therate of uptake (nanomoles of substrate per milligram of dry cells perminute) versus substrate concentration. Insets show double recip-rocal plots the rate of uptake versus substrate concentration.Estimated kinetic parameters: lactose, Km 10 ± 3 mM, Vmax 8 ± 2;sucrose, Km 10 ± 3 mM, Vma,j 45 ± 10; maltose, Km 5 ± 1 mM, Vnax15 ± 2. Data were obtained from five to seven independent exper-iments with each substrate.

protoplast fractions incubated as described below with thevarious disaccharides. Glucose determinations were carriedout with glucose oxidase (EC 1.1.3.4) by the oxygen con-sumption method. Malate dehydrogenase (EC 1.1.1.37) wasdetermined as described by Johnson and Hatch (16).Hexokinase (EC 2.7.1.1) was nmeasured by the method ofBergmeyer (3), and protein was measured by the method ofBradford (4).

RESULTSGrowth of T. cutaneum on six different disaccharides. The

cells were able to grow in a mineral salt medium containing1 mg thiamnine hydrochloride per liter when 0.5% ofcellobiose, melibiose, sucrose, maltose, lactose, or trehalosewas supplied as the sole carbon source. The doubling timesfor growth on glucose, cellobiose, melibiose, sucrose,maltose, and lactose were 3.5 to 4 h; the growth on eachsugar reached a final optical density at 580 nm of about 1.6 to2.0. Growth on trehalose was slower, with a doubling time ofabout 10 h, and the optical density did not reach a plateaueven after 32 h.

TABLE 1. Competition between uptake systems fordisaccharides in washed cells of T. cutaneuma

Growth Uptake of K. Competing K,subsrate 14C-labeled (MM unlabeled (Msubstrate substrate (mM) substrate (mM)

Maltose Maltose 5Sucrose 10 Sucrose 10a-MG _b a-MG 8

Sucrose Sucrose 10Maltose 6 Maltose 20a-MG _b a-MG 8

Lactose Lactose 10Glucose 3 Glucose 4

Cellobiose 9a Conditions were as described in the legend to Fig. 1.b -, No uptake of [14C] a-MG.

Kinetics and specificity of uptake of 14C-labeled disaccha-rides by T. cutaneum. Of the disaccharides studied, threewere available in labeled form: [U-14C]sucrose, [U-14C]maltose, and D-glucose-[1-14C]lactose. Uptake of thesedisaccharides was measured in washed cells that had beengrown on the respective substrates. The uptake of all threedisaccharides was linear for about 3 min. Uptake rates wereestimated from '4C label incorporated after 1 min of incuba-tion (Fig. 1). The specificity of disaccharide uptake wastested by competition studies. Washed cells were incubatedwith 14C-labeled growth substrate in the presence of unla-beled glucose, maltose, lactose, melibiose, cellobiose,trehalose, or sucrose. The results indicated that sucrosecompetes with the uptake of maltose in maltose-grown cellsand that maltose competes with the uptake of sucrose insucrose-grown cells. The uptake of lactose was competi-tively inhibited by both glucose and cellobiose. Table 1shows the respective Kms and Kis. Incubation of cells grownon maltose or sucrose with 2 to 10 mM [14C]a-MG for 1 to 10min did not result in any incorporation of radioactivity intocells. However, when unlabeled a-MG was added togetherwith [14C]maltose it caused competitive inhibition of maltoseuptake (Table 1). These results suggest that a carrier, pre-sumably common to maltose and sucrose transport, can binda-MG but cannot transport it into the cells.

Effect of energy inhibitors on the uptake of maltose, lactose,

TABLE 2. Effect of energy inhibitors on the uptake of14C-labeled maltose, lactose, and sucrose in cells grown on these

substratesa

InhibitorConcnPreincubation Uptake (% of control) of:Inhibitor (M) tm(in(mM) time (mmn) Maltose Sucrose Lactose

Oligomycin 0.05 2 100 91 10015 89 100 60

CCCP 0.01 2 100 100 850.05 100 56 250.10 85 36 25

Monensin 0.10 100

Valinomycin 0.005b 100a After preincubation with inhibitor, 10 mM of the respective 14C-labeled

substrate was added. Incubation was for 1 min. The data represent meanvalues from two to four independent experiments with each inhibitor.

b Higher concentrations of valinomycin caused aggregation of cells.

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736 MORTBERG AND NEUJAHR

TABLE 3. Glycosidase activities liberated during protoplastformation in cells grown on lactose, melibiose, and cellobiose

Glycosidase Malate

Growth substrate Cell fraction activitya dehydrogenase(nmol/mg of (mU/mg ofcells) cells)

Lactose (0.5%) Supematantb 638 9First wash 185 4Second wash 137 9Lysed 100 237

protoplasts

Melibiose (0.5%) Supernatantb 827 0First wash 97 0Second wash 82 8Lysed 90 71

protoplasts

Cellobiose (0.5%) Supernatantb 1,088 0First wash 375 10Second wash 102 9Lysed 135 100

protoplastsa Measured as nanomoles of glucose liberated during 30 min of incubation

with 10mM of the respective disaccharide at 28°C in 1.2 M sorbitol containing20 mM MES (pH 6.0). During analysis of lysed protoplasts, sorbitol wasomitted. The calculations of enzymatic activities in each fraction are based onthe amounts (dry weight) of cells that were used for preparation ofprotoplasts.

b Solution used to induce formation of protoplasts.

and sucrose. Two types of inhibitors were studied: thoseaffecting the ATP level of the cells, e.g., oligomycin andthose affecting the ion gradients across the cell membrane,the ionophores. The ionophores used were CCCP for H+,monensin for Na+, valinomycin for K+, and gramicidin formonovalent cations in general (Table 2). Preincubation for 2min with oligomycin did not significantly affect the uptakerate of any of the tested substrates. Preincubation for 15 minwith oligomycin affected the uptake rate of lactose very littleand that of maltose even less. Preincubation for 2 min withCCCP inhibited the uptake of lactose and sucrose in cellsgrown on these respective substrates, indicating that theuptake of these disaccharides is dependent on a protongradient. Preincubation of maltose-grown cells with CCCP,

monensin, or valinomycin did not significantly affect theuptake of maltose (Table 2). Preincubation for 2 min withgramicidin (0.005 to 0.1 mM) or valinomycin in concentra-tions higher than 0.005 mM caused aggregation of the cells.Thus, our results do not give any indication that gradients ofany of the positive ions H+, K+, and Na+ is involved in theuptake of maltose in T. cutaneum.

Extracellular glycosidase activities. Washed intact cellswere incubated with the respective growth substrate, andglucose liberated to the supernatant was determined. Lac-tose-grown cells were also tested with the non-metabolizablechromogenic substrate analog o-nitrophenyl-f-D-galacto-pyranoside. Results of these experiments indicated thatmaltose, sucrose, and trehalose may be taken up as such,since no glucose liberation could be observed. In contrast,lactose- and cellobiose-grown cells gave indications of ex-tracellular hydrolysis of o-nitrophenyl-p-D-galacto-pyranoside and cellobiose, respectively (data not shown).

It is possible that the estimation of extracellular glycosi-dase activities for maltose, sucrose, lactose, trehalose, andmelibiose is hampered by the liberated glucose, if formed,being rapidly taken up by the cells. This was checked bydetermining glucose in supernatant from intact cells whenenergy-dependent uptake of monosaccharides was inhibitedby CCCP. Preincubation for 2 min with 0.1 mM CCCPresulted in measurable amounts of glucose in the supernatantafter incubation with lactose or melibiose but not afterincubation with maltose, sucrose, or trehalose (data notshown). These results gave further indication that lactoseand melibiose are hydrolyzed externally, whereas externalhydrolysis of maltose, sucrose, or trehalose is not likely.

Glycosidase activities liberated during protoplast formationin cells grown on lactose, melibiose, and cellobiose. Theliberation of glycosidase activity from lactose-, melibiose-,and cellobiose-grown cells was measured during solubiliza-tion of the cell wall by lytic enzymes under conditionspreserving intact protoplasts. Determination of the intracel-lular enzyme malate dehydrogenase served as control ofstability of the protoplasts (Table 3). For all three substratesmore than 90% of the total amount of the respectiveglycosidase activities was liberated from the cell after solu-bilization of the cell wall.Trapping by hexokinase of hexoses liberated from washed

lactose-grown cells during incubation with lactose. Hexoses

3500[*

U,)H-500z

250-

0-

C C+CG T T+0 0 L L G a W. ME+G MACMA0 S SK+ C

GROWTH SUBSTRATEFIG. 2. Glycosidase activities in T. cutaneum. Glycosidase activity was measured as nanomoles of glucose liberated to supernatant by 1

mg of cell protein when detergent permeabilized cells were incubated for 30 min with 5 mM of the respective disaccharides in 0.1 M MES(pH 6.0) at 28 or 37°C. The two temperatures gave identical results. Growth substrates: C, cellobiose; T, trehalose; L, lactose; ME, melibiose;MA, maltose; S, sucrose; G, glucose.

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T. CUTANEUM TRANSPORT AND HYDROLYSIS OF DISACCHARIDES 737

TABLE 4. Inducibility of disaccharide uptakea

Growth substrate '4C-labeled Uptake (nmol/mgdisaccharide of cells per min)

Maltose (0.5%) Maltose (5 mM) 4.5 + 1.3Glucose (2.0%) Maltose (5 mM) 3.6 + 0.9Sucrose (0.5%) Sucrose (10 mM) 33.5 ± 10.4Glucose (2.0%) Sucrose (10 mM) 26.7 ± 10.6Lactose (0.5%) Lactose (10 mM) 6.0 ± 3.5Glucose (2.0%) Lactose (10 mM) 1.2 ± 0.1Lactose (0.5%) plus Lactose (10 mM) 1.5 ± 0.1

glucose (2.0%)

a Conditions were as described in the legend to Fig. 1. The data representmean values obtained from two to six independent experiments with eachsubstrate.

liberated by external glycosidase activity were trapped byadding hexokinase to washed suspensions of lactose-growncells incubated with lactose. The reaction was coupled toglucose-6-phosphate dehydrogenase and followed in a spec-trophotometer at 340 nm (3). Samples of the same suspen-sion were also tested for uptake of [14C]lactose to comparethe amount of glucose liberated exogenously with theamount of 14C label taken up. The results showed thatincubation of cells with 20 mM lactose for 4 min resulted ina mean value of 10.2 nmol of glucose liberated per mg (dryweight) of cells. Uptake of 14C label under the same condi-tions was 7.6 nmol of lactose per mg (dry weight) of cells.These results together show that lactose, cellobiose, and

melibiose are hydrolyzed by cell wall-bound glycosidase(s),indicating that they may be hydrolyzed before entering theinterior of the cells.

Induction of disaccharide uptake and hydrolysis. Cells weregrown on 0.5% of the various disaccharides, on 2% glucose,or on 0.5% disaccharide in the presence of 2% glucose. Theuptake of 14C-labeled lactose, maltose, and sucrose bywashed cells was determined as described above (see Table4). Glycosidase activities for the six respective disaccharideswere determined in detergent-permeabilized cells. Such es-timation gives the total glycosidase activity for each sub-strate (Fig. 2). From these data we conclude that the uptakesystems and enzymes for hydrolysis of maltose and sucroseare constitutive, since they were not affected by the pres-ence of glucose during growth. In contrast, the rates ofuptake and hydrolysis of lactose were markedly repressedby glucose. Also the enzymes hydrolyzing melibiose,trehalose, and cellobiose seem to be induced by growth onthe respective substrates and repressed by glucose.

DISCUSSION

Specificity and kinetics of disaccharide uptake. Accordingto a review by Barnett (2), between 50 and 60% of 439investigated yeasts can utilize sucrose, maltose, trehalose,and cellobiose, whereas only 11 to 12% of them utilizemelibiose or lactose. Our results show that T. cutaneum canutilize all of these substrates; the growth on five of them iscomparable to the growth on glucose, and the growth on thesixth (trehalose) is considerably slower. The half-saturationconstants for uptake were 10 + 3 mM sucrose or lactose and5 ± 1 mM maltose. The corresponding parameters for S.cerevisiae are 2.5 mM maltose (29) and about 6 mM sucrose(28), which are of the same order of magnitude. In T.cutaneum, sucrose and maltose may share a common trans-port system or parts thereof. In S. cerevisiae, the uptake ofsucrose was inhibited by maltose or trehalose (28). In our

experiments with T. cutaneum, trehalose did not affect theuptake of sucrose or maltose. The uptake rate of lactose wasmarkedly lowered in the presence of cellobiose or glucose;the Km and Ki for glucose in lactose-grown cells were nearlyidentical. These results suggest an interaction between theuptake system(s) for glucose, cellobiose, and lactose, if suchseparate systems exist. Alternatively, the results could beexplained by hydrolytic splitting of either disaccharide out-side the cell membrane and competition for uptake betweenthe resulting glucose moieties.Are the disaccharides hydrolyzed before uptake? Hydroly-

sis of disaccharides is the first metabolic step in theirutilization. The hydrolysis of lactose, cellobiose, and meli-biose by cell wall-bound glycosidases in T. cutaneum indi-cates that these disaccharides are split before or in connec-tion with uptake, whereas maltose, sucrose, and trehaloseare taken up as such. This is in contrast to what has beenreported for certain other yeasts. Lactose is considered to behydrolytically split inside the cell in a variety of yeastspecies (2). Using recombinant DNA-techniques, Dicksonand Markin (8) have shown that there are at least sevengenes involved in lactose utilization by Kluyveromyceslactis. One of these codes for a carrier. In Rhodotorulaglutinis the uptake of sucrose and trehalose was shown toinvolve exoenzymes (15). However, in S. cerevisiae, su-crose (28), trehalose (19), and maltose (29) are activelytransported without molecular change. An externally locateda-galactosidase for hydrolysis of melibiose has been demon-strated in Saccharomyces carlsbergensis (6). Also inCandida wickerhamii, a P-glucosidase has been shown tohydrolyze cellobiose mainly on the cell surface (17, 18).Are the uptake and hydrolysis of disaccharides inducible or

constitutive? The constitutive nature of uptake and hydroly-sis of maltose in T. cutaneum differs from that in S. cerevi-siae, in which an ax-glucosidase and a transport system arecoordinately induced (6) with three genes, at least, involvedin the process (5). However, sucrose utilization in S. cere-visiae is constitutive (28). There is an inducible 3-galactosidase in K. lachis (7), as in T. cutaneum. The abilityto ferment melibiose is inducible in S. carlsbergensis (6). Aconstitutive ,-glucosidase was found in C. wickerhamii (9),but an inducible 3-glucosidase was found in another strain T.cutaneum (14). Thus, there is seemingly no taxonomicregularity with respect to transport and hydrolysis of disac-charides by yeasts.

Quite large differences between the glycosidase levelswere observed in detergent-permeabilized cells of T.cutaneum (Fig. 2), but not during protoplast formation(Table 3). This could be explained by the topography of thelocation of the glycosidase(s). The permeabilization proce-dure might have resulted in some glycosidases (e.g., that forcellobiose) becoming exposed more than others to the sub-strate. This could also explain that, in spite of the largedifferences in glycosidase activities of permeabilized cells(Fig. 2), the growth on the corresponding disaccharides wasof a similar order of magnitude.Energy requirement. Our results indicate that the uptake

of lactose and sucrose is driven by a proton gradient. Also inS. cerevisiae, the uptake of sucrose is driven by protonmotive force (28). A system which hydrolyzes cellobiose andtransports the resulting glucose by a glucose-proton symporthas been demonstrated in C. wickerhamii (32). A similarsystem may be operating in T. cutaneum for lactose (forcellobiose and melibiose as well?). Maltose uptake in T.cutaneum may be by facilitated diffusion (through a carriershared with sucrose), or it may be driven by gradients of

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738 MORTBERG AND NEUJAHR

some ions not tested here. T. cutaneum thus differs from S.cerevisiae, in which maltose uptake has been shown to beenergy dependent and driven by a proton gradient (29).

ACKNOWLEDGMENTS

We are greatly indebted to Otto Andresen of Novo Industri A/Sfor the generous gift of NovoZym 234.The National Swedish Board for Technical Development pro-

vided financial support.

LITERATURE CITED1. Anderson, J. I., and S. Dagley. 1980. Catabolism of aromatic

acids in Trichosporon cutaneum. J. Bacteriol. 141:534-543.2. Barnett, J. A. 1981. The utilization of disaccharides and some

other sugars by yeast. Adv. Carbohydr. Chem. Biochem.39:347-404.

3. Bergmeyer, H. U. 1983. Hexokinase from yeast, p. 222-223. InJ. Bergmeyer and M. Grabl (ed.), Methods of enzymatic anal-ysis, vol. 2. Samples, reagents, assessment of results. VerlagChemi, Weinheim.

4. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

5. Cohen, J. D., M. J. Goldenthal, B. Buchferer, and J. Marmur.1984. Mutational analysis of the MALl locus of Saccharomy-ces: indentification and functional characterization of threegenes. Mol. Gen. Genet. 196:208-216.

6. De La Fuente, G., and A. Sols. 1962. Transport of sugars inyeast. Mechanisms of utilization of disaccharides and relatedglycosides. Biochim. Biophys. Acta 56:49-62.

7. Dickson, R. C., L. R. Dickson, and J. S. Markin. 1979. Purifi-cation and properties of an inducible ,3-galactosidase isolatedfrom the yeast Kluyveromyces lactis. J. Bacteriol. 137:51-61.

8. Dickson, R. C., and J. S. Markin. 1978. Molecular cloning andexpression in E. coli of a yeast gene coding for 3-galactosidase.Cell 15:123-130.

9. Freer, S. N., and R. W. Detroy. 1985. Regulation of P-1,4-glucosidase expression by Candida wickerhamii. Appl.Environ. Microbiol. 50:152-159.

10. Gaal, A., and H. Y. Neujahr. 1979. Metabolism of phenol andresorcinol in Trichosporon cutaneum. J. Bacteriol. 137:13-21.

11. Gaal, A. and H. Y. Neujahr. 1981. Induction of phenol-metabolizing enzymes in Trichosporon cutaneum. Arch. Micro-biol. 130:54-58.

12. Gaal, A. B. and H. Y. Neujahr. 1980. Maleylacetate reductasefrom Trichosporon cutaneum. Biochem. J. 185:783-786.

13. Gaal, A. B., and H. Y. Neujahr. 1980. Cis-cis-muconate cyclasefrom Trichosporon cutaneum. Biochem. J. 191:37-43.

14. Hrmova, M., P. Biely, M. Vrsanska, and E. Petrakova. 1984.Induction of cellulose- and xylan-degrading enzyme complex inthe yeast Trichosporon cutaneum. Arch. Microbiol. 138:371-376.

15. Janda, S. 1974. Uptake of disaccharides by the aerobic yeastRhodotorula glutinis. Arch. Microbiol. 101:273-280.

16. Johnson, H. S. and M. D. Hatch. 1970. Properties and regulationof leaf nicotinamide-adenine dinucleotide phosphate malate

dehydrogenase and "malic" enzyme in plants with the C4-dicarboxylic acid pathway of photosynthesis. Biochem. J.199:273-280.

17. Kilian, S. G. , B. A. Prior, and P. M. Lategan. 1983. Diauxicutilization of glucose-cellobiose mixtures by Candidawickerhamii. Eur. J. Appl. Microbiol. Biotechnol. 18:369-373.

18. Kilian, S. G., B. A. Prior, H. J. Potgieter, and J. C. du Prez.1983. Utilization of glucose and cellobiose by Candidawickerhamii. Eur. J. Microbiol. Biotechnol. 17:281-286.

19. Kotyk, A., and D. Michaljanitov6. 1979. Uptake of trehalose bySaccharomyces cerevisiae. J. Gen. Microbiol. 110:323-332.

20. Marquardt, D. W. 1963. An algorithm for least squares estima-tion of nonlinear parameters. J. Soc. Ind. Appl. Math.11:431-441.

20a.Miozzari, G. F., P. Niederberger, and R. Hutler. 1978.Permeabilization of microorganisms by Triton X-100. Anal.Biochem. 90:220-233.

21. Mortberg, M., and H. Y. Neujahr. 1985. Uptake of phenol byTrichosporon cutaneum. J. Bacterol. 161:615-619.

22. Nash, J. C. 1979. Compact numerical methods for computers:linear algebra and function minimisation. Adam Hilger Ltd.,Bristol, United Kingdom.

23. Neujahr, H. Y. 1978. Degradation of phenols by yeast. Proc.Biochem. 13:3-7.

24. Neujahr, H. Y. and A. Gaal. 1973. Phenol hydroxylase fromyeast. Purification and properties of the enzyme fromTrichosporon cutaneum. Eur. J. Biochem. 35:386400.

25. Neujahr, H. Y., and J. M. Varga. 1970. Degradation of phenolsby intact cells and cell-free preparations of Trichosporoncutaneum. Eur. J. Biochem. 13:37-44.

26. Powlowski, J. B., and S. Dagley. 1985. 3-Ketoadipate pathway inTrichosporon cutaneum modified for methyl-substituted metab-olites. J. Bacteriol. 163:1126-1135.

27. Powlowski, J. B., J. Ingebrand, and S. Dagley. 1985. Enzymol-ogy of the ,B-ketoadipate pathway in Trichosporon cutaneum. J.Bacteriol. 163:1136-1141.

28. Santos, E., L. Rodriguez, V. M. Elorza, and R. Sentandreu.1982. Uptake of sucrose by Saccharomyces cerevisiae. Arch.Biochem. Biophys. 216:652-660.

29. Serrano, R. 1977. Energy requirements for maltose transport inyeast. Eur. J. Biochem. 80:97-102.

30. Sparnins, V. L., J. J. Anderson, J. Omans, and S. Dagley. 1978.Degradation of 4-hydroxyphenylacetic acid by Trichosporoncutaneum. J. Bacteriol. 136:449451.

31. Sparnins, V. L., D. G. Burbee, and S. Dagley. 1979. Catabolismof L-tyrosine in Trichosporon cutaneum. J. Bacteriol. 138:425-430.

32. Spencer-Martins, I., and N. van Uden. 1985. Cataboliteinterconversion of glucose transport systems in the yeastCandida wickerhamii. Biochim. Biophys. Acta 812:168-172.

33. Stephen, E. R., and A. Nasim. 1981. Production of protoplasts indifferent yeasts by mutanase. Can. J. Microbiol. 27:551-553.

34. Varga, J. M., and H. Y. Neujahr. 1970. Purification and Prop-erties of catechol 1.2-oxygenase from Trichosporon cutaneum.Eur. J. Biochem. 12:427-434.

35. Varga, J. M., and H. Y. Neujahr. 1970. Isolation from soil ofphenol utilizing organisms and metabolic studies on the pathwayof phenol degradation. Plant Soil. 33:565-571.

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