JOURNAL OF BACTERIOLOGY, July 1982, p. 68-760021-9193/82/070068-09$02.0 O

Vol. 151, No. 1

Plasmid-Mediated Uptake and Metabolism of Sucrose byEscherichia coli K-12

KURT SCHMID, MARGIT SCHUPFNER, AND RUDIGER SCHMITT*Lehrstuhl fur Genetik, Universitat Regensburg, D-8400 Regensburg, Federal Republic ofGermany

Received 7 December 1981/Accepted 12 March 1982

The conjugative plasmid pUR400 determines tetracycline resistance and en-ables cells of Escherichia coli K-12 to utilize sucrose as the sole carbon source.Three types of mutants affecting sucrose metabolism were derived from pUR400.One type lacked a specific transport system (srcA); another lacked sucrose--phosphate hydrolase (scrB); and the third, a regulatory mutant, expressed both ofthese functions constitutively (scrR). In a strain harboring pUR400, both transportand sucrose-6-phosphate hydrolase were inducible by fructose, sucrose, andraffinose; if a scrB mutant was used, fructose was the only inducer. These datasuggested that fructose or a derivative acted as an endogenous inducer. Sucrosetransport and sucrose-6-phosphate hydrolase were subject to catabolite repres-sion; these two functions were not expressed in an E. coli host (of pUR400)deficient in the adenosine 3',5'-phosphate receptor protein. Sucrose uptake(apparent Km = 10 ,uM) was dependent on the scrA gene product and on thephosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) of thehost. The product of sucrose uptake (via group translocation) was identified assucrose-6-phosphate, phosphorylated at C6 of the glucose moiety. Intracellularsucrose-6-phosphate hydrolase catalyzed the hydrolysis of sucrose-6-phosphate(Km = 0.17 mM), sucrose (Km = 60 mM), and raffinose (Km = 150 mM). Theactive enzyme was shown to be a dimer of M, 110,000.

Plasmids conferring to their hosts the abilityto utilize sucrose as the sole carbon source havebeen isolated from strains of Salmonella andEscherichia coli (13, 35, 41). Previous investiga-tors have concentrated on the epidemiology,transmissibility, and molecular nature of varioussucrose plasmids. However, little is knownabout the genetic determinants (scr genes) andthe plasmid-encoded functions involved in su-crose metabolism and about their regulation andinteraction with host metabolic enzymes.Known metabolic plasmids that mediate the

utilization of lactose (4) or raffinose (29, 31) byE. coli were found to encode specific transportand hydrolyzing enzymes responsible for the"peripheral" metabolism of these sugars. Toanalyze the role played by plasmid-encodedfunctions in the metabolism of sucrose, we usedthe 52-megadalton conjugative plasmid scr-53(41) in the present study. To avoid confusionwith symbols for genetic loci that control theutilization of sucrose and in accordance with theproposed plasmid nomenclature (21), we willrefer to the plasmid as pUR400. This articledescribes (i) the isolation of scr mutants, (ii) thecharacterization of plasmid- and host-deter-mined functions required for sucrose utilization,and (iii) the identification of sucrose-6-phos-phate as the primary product of the phospho-

enolpyruvate (PEP)-dependent sucrose trans-port.

MATERIALS AND METHODSChemicals. [14C]glucose, [(4C]glucose-6phosphate,

[14CJfructose, [3H]raffinose, and [U-14C]sucrose werepurchased from NEN Chemicals GmbH (Dreieichen-hain) and [(4Cjlactose was purchased from AmershamBuchler (Braunschweig). [3H]melibiose was preparedfrom [3H]raffinose by hydrolysis with commercial in-vertase (Serva, Heidelberg) and subsequently purifiedby gel ifitration on Sephadex G-15. N-acetylglucosa-mine, streptozotocin, PEP, cAMP, and glucose-6-phosphate were obtained from Sigma Chemical Co.(Munchen); ovalbumin, aldolase, catalase, E. coliRNA polymerase, bovine serum albumin, trypsin in-hibitor, NADP, and tetracycline hydrochloride wereobtained from Boehringer (Mannheim), and N-methyl-N'-nitro-N-nitrosoguanidine were obtained fromServa. All other chemicals were purchased from E.Merck AG (Darmstadt).

Strains, plasmids, and mating procedure. Table 1lists the derivatives of E. coli K-12 and plasmids used.For conjugal transfer of sucrose plasmids, 0.5-mlportions of parental broth cultures (5 x 108 cells/ml)were mixed, mated for at least 2 h, diluted appropriate-ly, and spread on selective agar media. Scr+ exconju-gants were selected on minimal agar plates (30) con-taining 0.2% sucrose or on complete medium (39)supplemented with tetracycline (10 pLg/ml). Selectionfor tetracycline resistance was based on the findingthat pUR400 contains a tetracycline resistance deter-

68

PLASMID-MEDIATED SUCROSE METABOLISM IN E. COLI 69

minant in addition to sucrose markers (K. Schmid, J.Altenbuchner, and R. Schmitt, unpublished data).Donor cells were counterselected either by the omis-sion of required amino acids from minimal medium orby supplementing complete medium with streptomy-cin (200 ,ug/ml). Transduction with phage P1 followedthe procedure of Rothman (26).

Selection of scr mutants by streptozotozin treatment.Exponentially growing cells of DS402(pUR400) wereharvested, suspended in minimal medium without car-bon source at 109 cells/ml, and treated with nitroso-guanidine (100 Fg/ml) at 3rC for 30 min. The mutagenwas removed by centrifugation, and cells were trans-ferred to minimal medium supplemented with N-ace-tylglucosamine to allow for segregation and for induc-tion of the streptozotocin uptake system (14). After 16h of incubation at 37°C, cells were harvested, suspend-ed at 101 cells/ml in minimal medium supplementedwith 0.2% sucrose, and once more incubated at 37°C.Streptozotocin was added at a concentration of 50 ,ug/ml after one cell division, and growth was allowed toproceed for 3 h. This procedure selects for non-growing cells and increases the proportion of mutantsunable to utilize sucrose as a carbon source. Cellswere collected, washed, and grown overnight in mini-mal glucose medium. ScrC colonies were identified onMacConkey indicator plates (19) supplemented with1% sucrose instead of lactose.

Synthesis and Isolation of sucrose-6-phosphate. Su-crose-6-phosphate was prepared by incubating a cellsuspension of fructose-induced strain DS420 [about101 cells in 100 ml of 100 mM bis(2-hydroxymethyl)-imino-tris(hydroxymethyl)methane (Sigma), pH 7.0]with 10 ml of [14C]sucrose (9 mM; 1.1 mCi/mmol) at37°C for 10 min. Cells were sedimented at 23,000 x gfor 10 min, suspended in 100 ml of distilled water, andboiled for 5 min. After removal of cell debris bycentrifugation at 23,000 x g for 15 min, the clearsupernatant was concentrated to 4 ml under vacuum,using P205 as a desiccant. The concentrated extract

was fractionated on Sephadex G-15, using distilledwater for elution, and fractions containing 14C-labeledmaterial were pooled and again concentrated undervacuum to 200 pl (final volume). [14C]sucrose-6-phos-phate was separated from contaminating material bythin-layer chromatography on DC-cellulose and moni-tored by autoradiography (see below), isolated fromthe plate together with the adsorbant, and eluted withdistilled water. After a final concentration in vacuo, 1ml of a 1.7 mM solution of [14C]sucrose-6-phosphatewas recovered, corresponding to 2% final yield.Throughout the procedure, sucrose-6-phosphate wasmonitored by determining the radioactivity.Enzyme assays. Sucrose-6-phosphate hydrolase ac-

tivity was determined in bacterial cells exponentiallygrown in minimal glycerol medium with or withoutinducer (0.2%) treated with toluene (2 drops/5 x 108

cells at 25°C for 10 min). Alternatively, crude extractsfrom cell suspensions passed through a French pres-sure cell at 15,000 lb/in2 and freed from debris bycentrifugation (37,000 x g for 15 min) were used.Suspensions in 100 mM phosphate buffer, pH 6.6,were incubated with 50 mM sucrose at 25°C for 30 min.Enzymatic cleavage of sucrose was stopped by heatingthe reaction mixture at 100°C for 1 min. After removalof denatured protein by centrifugation, the liberatedglucose was determined in 0.1-ml samples by using acommercially available test solution (Merck) contain-ing glucose dehydrogenase (5.2 U/ml), mutarotase(0.11 U/ml), and NAD (1.1 mM) in phosphate buffer(12 mM, pH 7.6) and following the extinction at 365nm. The reaction was complete after 20 min at 25°C.Activities of sucrose-6-phosphate hydrolase are ex-pressed as nanomoles of glucose (or glucose-6-phos-phate) per minute per milligram of protein (1 mg ofprotein corresponds to 4 x 109 cells).The hydrolysis of sucrose-6-phosphate was deter-

mined by the amount of glucose-6phosphate released,using the glucose-6-phosphate dehydrogenase- andNADP-coupled assay (37). Contaminating glucose-6-

TABLE 1. Bacterial strains and plasmidsStrain Plasmid Plasmid markers' Chromosomal markers Source

WR3026 Scr+ pUR400 Scr+ Tcr lac thi his ile val met arg L. S. Baron (41)rpsL

K-12 Wild type E. LederbergCA8404 cya crp-45 rpsL 28; obtained from J. LengelerL191 lacY galT,K malA J. Lengeler

mtI(PO)SOgut(PO)SO gat(PO)50 thiargG his metB ptsl,HrpsL

DS402 pUR400 Scr+ Tcr Prototroph WR3026 Scr+ x K-12DS402-1 pUR400 Scr+ Tcr crp-45 rpsL P1 transduction of crp-4S rpsL

from CA8404 into DS402DS405 pUR401 scrA(EIIwr) Tcr Prototroph This paperDS409 pUR404 scrR(Scrns`) Tcr Prototroph This paperDS415 pUR400 Scr+ Tcr ptsl,H rpsL DS402 x L191DS420 pUR406 scrB (Hyd-) Tcr Prototroph This paper

a The symbol scr stands for plasmid-encoded mutant loci that control the utilization of sucrose. "Scr" and"Hyd" denote phenotypic traits concerning the ability to utilize sucrose and to synthesize hydrolase,respectively; "const" denotes constitutive expression of sucrose functions, and "Tcr" denotes tetracyclineresistance. Other genetic symbols are those of Bachmann and Low (2).

VOL. 151, 1982

70 SCHMID, SCHUPFNER, AND SCHMITT

phosphate (approximately 4% in our preparation) wasconsumed by preincubation of the reagents before thereaction was initiated by adding the extract containingsucrose-6-phosphate hydrolase. Glucose-6phosphatewas assayed in 100 mM Tris, pH 7.6, by incubation at20°C with 1 U of glucose-6phosphate dehydrogenase(Sigma) in the presence of 5 mM NADP. The amountof NADPH generated by the reduction of glucose-6-phosphate was determined by the extinction at 340nm.Transport assay. The substrate, [U-_4C]sucrose,

was freed from contaminating sugars by gel filtrationon Sephadex G-15, and its purity was tested by thin-layer chromatography on DC-cellulose. Bacteria weregrown at 37°C in minimal medium supplemented with0.2% of the carbon source indicated to a density of 1.5x 109 cells/ml. Mutants unable to utilize the inducingsugar as carbon source were grown in Luria broth (10 gof tryptone, 5 g of yeast extract, and 5 g of sodiumchloride per liter). Cells were harvested at roomtemperature, washed three times, and suspended in100 mM phosphate buffer, pH 6.6, to a final density of5 x 10' cells/ml. Routinely, 0.6 ml of cell suspensionwas added to 100 p,l of [14C]sucrose, rapidly mixed,and incubated at 25°C. Samples (0.2 ml) were takenafter 20, 40, and 60 s, collected on cellulose nitratedisks (0.6-p.m pore size; Sartorius, Gottingen) byfiltration, and washed twice with 1 ml of buffer. Filterswere dried at 80°C for 30 min, and their radioactivitywas determined in a Beckman LS-335 scintillationcounter, using 5 ml of Permablend II (Packard Instru-ments GmbH, Frankfurt). The nonspecific adsorptionof radioactivity was determined by using a plasmid-free strain deficient in sucrose transport. Transportactivities are expressed in nanomoles per minute permilligram of protein.

In vitro phosphorylation of [14C]sucrose by EIIW.The preparation of cell-free extracts and the assay ofsucrose-specific enzyme II (EII01) were essentiallyconducted as described by Lengeler and Lin (15).Extracts of wild-type E. coli K-12 served as the sourceof PEP-dependent sugar:phosphotransferase system(PTS) enzymes I (EI) and HPr. Extracts containingmembrane vesicles of fructose-induced cells of DS420provided the specific EIIl". Extracts (20 to 30 mg ofprotein/ml) were obtained by sonication (3 min at 75W, 4°C; Branson Sonifier; Branson Instruments Co.,Stamford, Conn.) and subsequent centrifugation(37,000 x g for 20 min). The reaction mixture consist-ed of 20 PIu of PEP (50 mM), 20 PIu of MgCJ2 (0.5 mM),40 pl of Tris (100 mM, pH 7.6), 100 PI1 of E. coli K-12extract, and 20 PIu of ['4C]sucrose (4.3 mCi/mmol; 0.36mM); 20 pl of strain DS420 extract was added to startthe reaction (25°C). After 1, 5, and 10 min, 50-Plsamples were transferred to DEAE filters (DE81;Whatman), placed into 80%o ethanol for 10 min, andwashed three times with distilled water. Filters weredried at 80°C for 30 min, and the radioactivity retainedwas determined.

Thin-layer chromatography. Ten microliters of 14C-labeled carbohydrate was separated by cellulose thin-layer chromatography (DC-cellulose; Merck), using amixture of n-butanol, acetic acid, and water (5:3:2).Plates were dried, and autoradiographs were taken in aspark chamber (Beta-Kamera LB29OB; Berthold,Wildbad).

RESULTSGeneral properties of the pUR400-encoded su-

crose system. The conjugative sucrose plasmidpUR400 enables cells of E. coli K-12 to utilizesucrose as the sole carbon source (41). In addi-tion to the scr-specific genetic loci, we havedetected a plasmid-borne tetracycline resistancedeterminant. Preliminary experiments indicatedthat at least two inducible plasmid-encodedfunctions were involved in sucrose metabolism:(i) a sucrose-specific transport system; and (ii) ahydrolyzing enzyme, P-D-fructofuranoside fruc-tohydrolase (EC 3.2.1.26), referred to as su-crose-6-phosphate hydrolase (3, 37). These func-tions were not detectable in the plasmid-free E.coli K-12. The plasmid-containing derivativeDS402(pUR400) expressed low levels of sucrosetransport and sucrose-6-phosphate hydrolase ifgrown on glycerol (no induction). If grown oninducing substrates such as fructose, sucrose, orraffinose, both sucrose transport and hydrolaseactivities increased up to 70-fold (Table 2). In-duction by raffinose was dependent on a fullyinduced or constitutively expressed lac operon,indicating that raffinose required lacY-encodedtransport to enter the cells (S. Schaeffler, Bac-teriol. Proc., 1967, p. 54; 28). Glucose, galac-tose, lactose, or melibiose was not an inducer ofsucrose metabolic enzymes. The simultaneousinduction of sucrose transport and sucrose-6-phosphate hydrolase by three different carbohy-drates suggested that the two functions wereunder common genetic control. This notion wassupported by the properties of a constitutivemutant (DS409), which expressed high-level ac-tivities of sucrose transport and hydrolase with-out induction (Table 2). In sucrose-induced cellsof DS402, the expression of both sucrose trans-port and hydrolase was about 12-fold reduced ifglucose had been added to the growth medium.This together with the observation that additionof cAMP to the culture led to maximal expres-sion suggested that the sucrose system was alsosubject to catabolite repression (20).

Isolation and analysis of scr mutants. Screeningof mutagenized and streptozotocin-treated cul-tures of DS402(pUR400) on MacConkey su-crose plates produced two classes of mutantsunable to metabolize sucrose. One class,DS405(pUR401), was deficient in sucrose trans-port (scrA), and the other class, DS420(pUR406),was deficient in sucrose-6-phosphate hydrolase(scrB). The mutational blocks were identified bydetermining sucrose transport and sucrose-6phosphate hydrolase activities in induced andnoninduced cells (Table 2).The glucose effect exerted on sucrose utiliza-

tion by DS402 (Table 2) was used to selectconstitutive mutants. Since systems sensitive to

J. BACTERIOL.

PLASMID-MEDIATED SUCROSE METABOLISM IN E. COLI 71

catabolite repression depend on cAMP andcAMP receptor protein (CRP) for full expression(23, 42), a deficiency in either of these com-pounds leads to a negative phenotype. If crp45,a deletion in the structural gene of CRP (27) andcotransducible with streptomycin resistance (2),was transferred into DS402 by P1 transduction,Scr- streptomycin-resistant transductants wereisolated, which formed pale colonies on Mac-

TABLE 2. Characterization of scr mutants byactivities of sucrose transport and sucrose-6

phosphate hydrolase

Uptake of Sucrose-6-sursb phosphate

Straina Inducing substrate (pmol/min (hydolsme(0.2%) per mg of n

protein) per mg ofprotein)

K-12 None <1 <1Fructose <1 <1

DS402 None 5 4(scr+) Fructose 320 140

Sucrose 280 190Sucrose + glu- 24 14cose

Sucrose + cAMP 450 360(6 mM)

Raffinosec 380 240DS405 None <1 6

(scrA) Fructose <1 170Sucrose <1 10Raffinosec <1 120

DS420 None 5 <1(scrB) Fructose 360 <1

Sucrose 4 <1Raffinosec 5 <1

DS409 None 1,230 620(scrR) Sucrose 250 90

Glucose 220 90Sucrose + cAMP 1,100 500

(6 mM)DS415 None <1 <1

(WtslI) Fructose <1 20Fructose + cAMP <1 150

(6 mM)

a Cells were grown at 37°C in minimal medium (30)containing 0.2% glycerol (no inducer). Cells of the ptsmutant DS415 were grown in Luria broth. Inducersand cAMP were added as indicated. Activities wereassayed in exponentially growing cells at maximalinduction. Values represent averages of three experi-ments, the deviations being +50%0.

b Uptake was determined at 60 nM [14C]sucrose(673 rmCi/mmol).

C Raffinose enters the cells via lacY-encoded per-mease fully induced by 100 F.M isopropylthio-galactoside.

Conkey sucrose plates. Spontaneous revertantsdetectable by their pink color were isolated after3 days at 37°C. When their plasmids were trans-ferred to the crp+ host strain K-12, resultingtransconjugants [e.g., DS409(pUR404)] showedhigh-level constitutive expression of sucrosetransport and sucrose-6-phosphate hydrolase.Their expression was still subject to cataboliterepression by sucrose and glucose, which wasrelieved by cAMP (Table 2), indicating that themutation leading to constitutivity did not pro-duce a CRP-independent scr promoter (5). Con-ceivably, the mutant locus resides in a plasmid-borne regulatory gene, designated scrR.An analysis of scrA and scrB mutants revealed

certain features of the sucrose system concern-ing inducer and substrate specificities. In thehydrolase-deficient strain DS420, sucrose trans-port was inducible by fructose, but not by su-crose and raffinose, suggesting that fructose or aderivative (fructose phosphate?) was the actualinducer of the scr genes. Hence, sucrose andraffinose required hydrolysis by sucrose-6-phos-phate hydrolase to become inducers. In thetransport mutant DS405, hydrolase was induc-ible by raffinose and fructose, but not by su-crose. Whereas fructose (6) and raffinose(Schaeffier, Bacteriol. Proc., 1967, p. 54; 28)could enter the cells by host transport systems,sucrose could not. The plasmid-encoded prod-uct of scrA was the only uptake system specificfor sucrose and was therefore absolutely re-quired for the utilization of this sugar by E. coliK-12. As shown below, sucrose transport wasdependent on the PTS of the host bacterium (8,11). The scrA gene encodes the membrane-bound EIIScr of the PTS.

Properties of sucrose transport. Transport ac-tivities were determined by the initial rates ofuptake, using [14C]sucrose as the substrate. Thetransport activity of fructose-induced DS402(pUR400) was 0.4 nmol/min per mg of protein(Fig. IA); it was low in uninduced DS402, and itwas not detectable in the plasmid-free E. coli K-12. The constitutive mutant DS409 exhibited athreefold higher initial uptake (Fig. 1B) thaninduced cells of DS402 (Fig. 1A). However, therate of sucrose accumulation decreased rapidlyin the former and reached a plateau after 60 s. Atthis level, the influx of [14C]sucrose and theefflux of 14C-labeled metabolites are thought tobe at equilibrium, an interpretation supported bythe hydrolase-deficient mutant DS420 showinglinear uptake kinetics for at least 80 s (Fig. 1B).Linearity was extended in DS420 to severalminutes by increasing the external substrateconcentration (data not shown), which indicatedthat substrate was continuously accumulated (inthe form of sucrose phosphate), but could not bereleased from a strain unable to further metabo-

VOL. 151, 1982

72 SCHMID, SCHUPFNER, AND SCHMITT

106-

0.3

CL

~~~~~~~~~~~~~~0.40.2

0 0

0 20 40 60 80 100 120 0 20 40 60 80 100 120

Time (sec) Time (sec)

FIG. 1. Sucrose uptake by the plasmid-encodedsucrose transport system. ["Clsucrose (673 mCi/mmol; 0.130 ,M) was used. (A) Sucrose uptake by E.coli K-12 (Scr-) grown on 0.2% fructose (O) and byDS402 (Scr+) grown on 0.2% glycerol (0; no induc-tion) or 0.2% fructose (0; induction). (B) Sucroseuptake by DS409 (scrR) grown on 0.2% glycerol (0)and by DS420 (scrB) grown on 0.2% fructose (0).

lize it. Assuming a cell water volume of 1.3 x10-12 ml per cell (40), we calculated that theintracellular substrate concentration of mutantstrain DS420 was 700-fold above the externalconcentration after 1 min (Fig. 1B).

Sucrose was the preferred substrate of thesucrose transport system. Glucose or fructose ata 10 mM concentration (1,000-fold excess) led toonly 50%o inhibition of sucrose uptake. The highconcentrations of inhibitor needed suggestedthat both glucose and fructose were poor sub-strates. No uptake of [3H]raffinose, [3H]meli-biose, and [l Cllactose could be detected inappropriate mutant hosts of pUR404 lackingmelibiose (30) and lactose (9) permease (data notshown).

Sucrose transport is PTS dependent. WhenpUR400 was conjugally transferred into the ptsmutant L191 (by selection for plasmid-bornetetracycline resistance), the resulting transcon-jugant DS415(pUR400) had a Scr- phenotype.Upon induction with fructose, the strain showedno detectable sucrose uptake, but sucrose-6-phosphate hydrolase activity. The latter wasabout sevenfold reduced relative to the fructose-induced wild-type DS402 (Table 2). The lowhydrolase activity in DS415 has been ascribed toreduced levels of cAMP observed in PTS-strains (22, 24). The inhibition of plasmid scrBgene expression owing to catabolite repression(23, 42) was released by the addition of 6 mMcAMP to growing cells of DS415, which in-creased the hydrolase activity nearly to the levelof wild-type cells (Table 2). However, cAMP didnot at all stimulate sucrose uptake, suggestingthat an intact PTS was required for the transportof sucrose.

The presence of an intact scrA gene in the ptsmutant DS415 has been demonstrated by out-crossing and expression in a PTS+ strain.Expression of the scrA gene in the PTS- back-ground of strain L191 containing pUR404 (scrR)grown in Luria broth was shown after in vitroreconstitution (15) of membrane vesicles of thisstrain with cell extracts of E. coli K-12 contain-ing the PTS proteins (Lengeler, personal com-munication). It has been concluded that theplasmid-borne scrA gene encodes a membrane-bound EIISC" acting in concert with the solublePTS functions, El and HPr, provided by the host(11). As shown in the accompanying paper (16),sucrose uptake is exceptional in requiring anadditional host function, termed enzyme IIIg1c,scr(EIII5lcscr). Since PTS-mediated transport in-volves vectorial phosphorylation (8, 11), sucroseuptake should lead to the accumulation of su-crose phosphate.

In vitro phosphorylation of sucrose. Substrate-specific phosphorylation can be assayed in vitroif cell extracts containing the soluble compo-nents (EI and HPr) and membrane vesiclescontaining the specific transport protein (EII)are used (15). Typically, this phosphorylation isdependent on PEP. The phosphorylation of su-crose in extracts of E. coli K-12 (containingexcess El, HPr, and EIIIgsc,scr; 15) mixed withmembrane vesicles of fructose-induced DS420(containing EIIScr) strictly depended on the pres-ence of PEP in the mixture (Fig. 2). PEP couldnot be replaced by ATP.

Sucrose was phosphorylated by the systemwith a Km of 12 ± 5 ,uM (Fig. 3). This valuecompares with an apparent Km of 10 ± 3 ,uM ofin vivo transport (Fig. 3), cafculated from theinitial rate of sucrose uptake by fructose-in-duced cells of DS420 (Fig. 1A). This closesimilarity of values suggested that we did, infact, measure the same reaction in vivo and invitro. The observed specificities of in vitro phos-phorylation corroborated this statement: raffi-nose, melibiose, lactose, and lactulose (4-O-p-D-galactosyl-D-fructose) did not inhibit thereaction, and glucose and fructose led to 40%oinhibition if added at a 10 mM concentration(1,000-fold excess). Differences in Vmax values(for transport, Vmax = 100 nmol/min per mg; forphosphorylation, V.max = 0.5 nmol/min per mg;Fig. 3) were ascribed to the low activity of the invitro system. In our hands, the latter could notbe improved by other methods of cell extractionsuch as toluene treatment (7). Conceivably,EIIScr itself or its interaction with host functionswas especially sensitive to extraction, resultingin the low specific activity observed in vitro.

Identification of sucrose-6-phosphate as the pri-mary metabolite accumulated by Scr+ cells. Fruc-tose-induced cells of the scrB mutant DS420

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PLASMID-MEDIATED SUCROSE METABOLISM IN E. COLI 73

were incubated with [U-14C]sucrose and subse-quently extracted with boiling water (see Materi-als and Methods). Thin-layer chromatography ofextracts revealed a slowly migrating radioactivecompound different from sucrose (not shown).The new compound was isolated from platesand, upon hydrolysis by sucrose-6-phosphatehydrolase (from DS409) or boiling hydrochloricacid, produced two species comigrating withfructose and glucose-6-phosphate, respectively,in cellulose thin-layer chromatography (Fig. 4).The putative glucose-6-phosphate was isolatedfrom the chromatogram, and its chemical naturewas verified by the reaction with glucose-6-phosphate dehydrogenase. Based on the specificradioactivity of the substrate and the turnover ofNADP, more than 90% (9.6 nmol) of the isolatedcompound had been converted (to 6-phospho-gluconate) after 15 min by the highly specificenzyme reaction. From the assignment of glu-cose-6-phosphate, we deduced that the immedi-ate product of PTS-dependent sucrose uptakewas sucrose-6-phosphate phosphorylated at glu-cose-C6.

Sucrose-6-phosphate hydrolase: enzymatic ac-tivity, substrate specificity, and molecular weight.The enzyme hydrolyzing sucrose-6-phosphate islocated intracellularly; no activity was found inthe membrane fraction or in periplasmic cold-shock preparations. The hydrolysis of sucrose-

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FIG. 3. Lineweaver-Burk plot of initial velocitiesof sucrose uptake in vivo (0) and sucrose phosphor-ylation in vitro (0). To cells of DS420 grown on 0.2%fructose, [14C]sucrose was added at concentrationsranging between 1.9 and 14 FM, and samples weretaken 10 s after the addition of substrate. The phos-phorylation of [14C]sucrose was assayed at substrateconcentrations from 5 to 100 ,uM, and the reaction wasstopped after 3 min. 1/v is expressed in nanomoles perminute per milligram of protein.

6-phosphate and of sucrose was optimal in 100mM phosphate at pH 6.6. The enzyme was stablefor at least 2 h at 37°C or for 3 days at 4°C.Enzyme preparations showed a rapid loss ofactivity at low ionic strength. After 24 h ofdialysis against 10 mM phosphate buffer, lessthan 1% of the initial activity was recovered.

- Fructose- Glucose

5

Time (min)FIG. 2. Sucrose phosphorylation in vitro and de-

pendence on PEP. Soluble extract containing El andHPr from strain K-12 and membranes of DS420 weredialyzed against 100 mM Tris, pH 7.0, before the assay(to remove PEP and ATP; see Materials and Meth-ods). Addition of 2.5 mM ATP (0) or 2.5 mM PEP (0)to the reaction mixture.

t ... - Glucose-6PStart-

a b c d e

FIG. 4. Autoradiogram of 14C-labeled sugars sepa-rated on a thin-layer cellulose plate. The originalproduct of sucrose transport was extracted from fruc-tose-induced cells of DS420 incubated with [14C]su-crose (see Materials and Methods). Before chromatog-raphy, all samples were boiled in 0.1 N HCI for 10 min(to hydrolyze disaccharides) and then neutralized withNaOH. Lane a, Acid-hydrolyzed [14C]sucrose; lane b,acid-hydrolyzed PTS reaction product; lane c, [14C]glu-cose; lane d, [14C]fructose; lane e, [14C]glucose-6-phosphate.

VOL. 151, 1982

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74 SCHMID, SCHUPFNER, ANDSCHMIJT

Additions such as dithiothreitol, metal ions,glycerol, or sucrose did not significantly stabi-lize the enzyme. Heavy losses in enzymaticactivity occurred when purification by conven-tional methods was attempted. Therefore, cell-free extracts of E. coli DS409 were routinelyused without further fractionation to assay theenzyme.

Sucrose-6-phosphate hydrolase catalyzed thehydrolysis of 1-D-fructosides, such as sucrose-6-phosphate, sucrose, and raffinose. The Michae-lis constants for these substrates were 0.17 ±0.05, 60 ± 10, and 150 ± 10 mM, respectively.The results suggested that sucrose-6-phosphatewas the essential substrate of the hydrolyzingenzyme. a-D-Glucosides such as p-nitrophenyl-a-D-glucoside and galactosides such as meli-biose and lactose were not hydrolyzed. Thisspecificity classifies the enzyme as a 1-D-fructo-furanoside fructohydrolase (EC 3.2.1.26).

Enzymatically active sucrose-6-phosphate hy-drolase had an Mr of approximately 110,000 asdetermined by gel filtration on Sephadex G-200,using ovalbumin, aldolase, and catalase for cali-bration (1; data not shown). A partially purifiedenzyme preparation (using ammonium sulfateprecipitation and fractionation on octyl-Sephar-ose CL-4B and on hydroxyapatite) was analyzedby sodium dodecyl sulfate-gel electrophoresis,using trypsin inhibitor, bovine serum albumin,and RNA polymerase for calibration (32). Themobility of the major protein band correspondedto Mr 55,000 (data not shown), suggesting thatthe active enzyme was a 110,000-dalton dimerconsisting of identical subunits.

DISCUSSIONPlasmid pUR400 mediates the "peripheral"

metabolism of sucrose in E. coli K-12 by furnish-ing the sucrose-specific, membrane-bound com-pound EIISC' of the PTS and the hydrolyzingenzyme sucrose-6-phosphate hydrolase. Thehost contributes the general PTS proteins, in-cluding El and HPr (8, 11), and, as shown in theaccompanying paper (16), an additional factor,E11191C,scr, which is required for the activity ofthe sucrose transport system. Figure 5 illustratesthe interplay of plasmid- and chromosome-en-coded functions as currently understood. Thisconcept is a result of the genetic and biochemi-cal studies presented in this paper.The analysis of scr mutants revealed the exis-

tence of at least three plasmid-encoded geneticloci distinguished by the biochemical alterationsthey produced. These pertain to sucrose trans-port (scrA, strain DS405), sucrose-6-phosphatehydrolase (scrB, strain DS420), and constitutiveexpression of the two functions (scrR, strainDS409). Regulatory mutants such as DS409 sug-gest that the scr functions are under common

genetic control, a notion also supported by thesimultaneous induction of sucrose transport andsucrose-6-phosphate hydrolase by fructose, su-crose, and raffinose (DS402; Table 2). Resultsobtained with the hydrolase-deficient mutantDS420 revealed that neither sucrose nor raffi-nose, but only fructose was capable of inducingthe scr system.

This is reminiscent of the lac system, whichrequires the conversion of lactose into allolac-tose by 0-galactosidase for induction (10). Themechanism may ensure that the disaccharidecan be broken down by the cell before wastingenergy for its uptake. With the available data wecannot determine whether fructose or a metabo-lite of fructose functions as the endogenousinducer. Externally supplied fructose is translo-cated (predominantly) via two fructose PTSs asfructose-i-phosphate and fructose-6-phosphate,whereas the primary intracellular product ofsucrose-6-phosphate hydrolysis is fructose (Fig.5), which is converted to fructose-6-phosphateby an ATP-dependent kinase (6). In an analogyto the sucrose system, Lengeler and Steinberger(17) observed that the PTS-dependent mannitoland glucitol systems were inducible by the intra-cellular unphosphorylated substrates. These au-thors favor a model in which the unphosphory-lated ElI complex of the PTS functions as abidirectional carrier for the free substrate,whereas the phosphorylated complex catalyzesvectorial phosphorylation. This concept allowsfor minor intracellular concentrations of unphos-phorylated substrate sufficient for induction. Italso accounts for our observation that the ptsmutant DS415 was inducible by fructose (Table2). The nature of the endogenous inducer of the

SCR

FIG. 5. Proposed gene-enzyme relationships in thesucrose metabolism of E. coli DS402. Plasmid pUR400(left) and a portion of the host chromosome (right) withrelevant genetic loci are shown together with plasmid-mediated steps in sucrose metabolism (SCR-6-P, su-crose-6-phosphate; GLC-6-P, glucose-6-phosphate;FRU, fructose; FRU-P, fructose phosphate). GeneralPTS proteins refer to EI, HPr, and EIII'Ocr (16).

J. BACTERIOL.

PLASMID-MEDIATED SUCROSE METABOLISM IN E. COLI 75

sucrose system can unequivocally be deter-mined in an in vitro system, which is currentlybeing developed.Our results are compatible with a plasmid-

encoded "scr operon" or "scr regulon" control-ling the synthesis of the specific transport com-ponent EIIScr and of sucrose-6-phosphatehydrolase. Although none of the mutants hasbeen shown to lie in the structural genes, newlyisolated polar insertions are in favor of an scroperon with the gene order scr(O,P) scrA scrB(K. Schmid, J. Altenbuchner, and R. Schmitt, tobe published). The data available are not suffi-cient for clearly defining the nature of the regula-tory mutant DS409. Since it was isolated as aScr+ revertant from a crp- background, it wasexpected to overcome catabolite repression.However, the constitutive expression of sucrosetransport and sucrose-6-phosphate hydrolase (inthe crp+ background of DS415) was sevenfolddecreased by sucrose or glucose, and the repres-sion was released by cAMP (Table 2). Instead ofthe expected CRP-independent promoter muta-tion (5, 42), the isolated sucrose-constitutivemutant DS409 (pUR404) is likely to represent aregulatory gene mutation, such as the araCc(activator gene constitutive) mutation of thearabinose system (33) or the lacI (repressornegative) mutation of the lactose system (9).Preliminary data on a newly isolated sucrose-constitutive mutant obtained after transposonmutagenesis favor negative control exerted bythe product of scrR on the expression of scrstructural genes (Schmid et al., to be published).The current concept, as illustrated in Fig. 5,involves a twofold control by (i) the scrR prod-uct (regulator) interacting with the scr operatorand with fructose or a derivative (inducer) and(ii) catabolite activation by the cAMP-CRP com-plex interacting with the scr promoter (5, 42).This model is presently being tested and refinedby experiments that include transposon muta-genesis, physical mapping, and complementa-tion.PEP-dependent EIIScr activity was demon-

strated by in vitro reconstitution (Fig. 2) and bythe inability of a pts mutant to transport sucrose(Table 2). The product of vectorial phosphoryla-tion was isolated and characterized as sucrose-6-phosphate by hydrolysis into fructose and glu-cose-6-phosphate (Fig. 4). The hydrolyzingenzyme, sucrose-6-phosphate hydrolase, is a110,000-dalton dimer which in molecular sizeand substrate specificity closely resembles an"invertase" encoded by the plasmid-borne raffi-nose operon (31). The possible relationship be-tween these hydrolases from different geneticsystems will be analyzed by serology and DNAhybridization.Known PTS sugars of E. coli were previously

restricted to monosaccharides and polyols (17).The scr system is novel in that it represents thefirst PTS-dependent disaccharide transportlinked to a plasmid. Other possible candidatesfor PTS-dependent disaccharide transport in E.coli are the bgl system, which controls theuptake and metabolism of P-glucosides such asarbutin, salicin (25), and possibly cellobiose, andthe trehalose system mentioned by Lengeler etal. (16). It is conceivable that the scr geneticsystem of pUR400 originated from another bac-terial species. Similar routes of sucrose metabo-lism have been described in the gram-positivebacteria Bacillus subtilis (18), Streptococcuslactis (12, 38), and Streptococcus mutans (3, 34,37). Though related in the overall reactionscheme and in the specificity of their sucrosefunctions, these systems are inducible by su-crose (and some analogs), but not by fructose.Moreover, the molecular weights reported forthe enzymes hydrolyzing sucrose-6-phosphatein S. lactis (Mr 28,000; 38) and S. mutans (Mr42,000; 3) are considerably smaller than Mr110,000 found for the pUR400-encoded dimericenzyme. A sucrose metabolic system similar tothe one described here has been recently identi-fied in the gram-negative bacterium Klebsiellapneumoniae (16), which therefore may be con-sidered a potential ancestor of the pUR400-encoded sucrose functions. The possible originof the scr genes and relationships among varioussucrose plasmids could be determined either bySouthern hybridization (36) or by the immuno-chemical cross-reaction of their hydrolases. Thelatter technique has been successfully applied toestablish relationships among the raffinose genesof 39 plasmids (28). Although various sucroseplasmids differ in size, transmissibility, and in-compatibility (13, 35, 41), their scr determinantsmay well be related. Experiments are in pro-gress to investigate whether inverted repeats orinsertion elements flanking the scr genes may beresponsible for their spread between plasmidsand chromosomes, as has been shown for plas-mid-borne lactose (4) and raffinose genes (31).

ACKNOWLEDGMENTSWe thank Joseph Lengeler for valuable comments on the

manuscript, Gerda Pensel for technical assistance, and HeleneBeier for artwork.

This investigation was supported by the Deutsche Fors-chungsgemeinschaft.

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