JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4288-42940021-9193/90/084288-07$02.00/0Copyright © 1990, American Society for Microbiology

Vol. 172, No. 8

A Novel Membrane-Associated Threonine Permease Encoded by thetdcC Gene of Escherichia coli

VENIL N. SUMANTRAN, HERBERT P. SCHWEIZER,t AND PRASANTA DATTA*Department ofBiological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-0606

Received 18 December 1989/Accepted 4 May 1990

A novel L-threonine transport system is induced in Escherichia coi cells when incubated in amino acid-richmedium under anaerobic conditions. Genetic and biochemical analyses with plasmids harboring mutatons inthe anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible forL-threonine uptake. Competition experiments revealed that the L-threoni transport system is also involved inL-serine uptake and is parially shared for L-leucine transport; L-alanine, L-valine, and L-sline did notaffect L-threonine uptake. Transport of L-threonine was inhibited by the respiratory chain II KCN andcarbonyl cyanide m-chlorophenylhydrazone and was Na+ independent. These results identify for the first timean E. coli gene encoding a permease specific for L-threouine-L-serine transport that is distinct from thepreviously described threonine-serine transport systems. A two-dimensional topological model predited fromthe amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integralmembrane protein with several membrane-spanning domains exhibiting a striking similarity with otherbacterial permeases.

When incubated anaerobically in tryptone yeast extract(TYE) medium, Escherichia coli synthesizes biodegradativethreonine dehydratase (EC 4.2.1.16) that catalyzes the de-hydration of L-threonine and L-serine to ammonia and to thecorresponding a-keto acids (32). Earlier, Hobert and Datta(18) described a synthetic medium (H4) which consists offour amino acids, threonine, serine, valine, and isoleucine,plus fumarate and cyclic AMP and supports high level ofenzyme production during anaerobiosis. Recently, the struc-tural gene for the dehydratase has been cloned on a 6.3-kilobase EcoRI DNA fragment (10, 13). A variety of exper-iments including DNA sequence analysis, deletion studies,minicell expression, and insertion mutagenesis (13, 26, 27)indicated that tdcB encoding threonine dehydratase is part ofthe tdcABC operon that harbors two additional genes, tdcAand tdcC (Fig. 1). In addition, genetic analysis of the clonedDNA revealed that efficient expression of the operon re-quires the product of a regulatory gene, tdcR, situatedupstream of the tdc promoter in opposite transcriptionalorientation (28).The enzymatic function of the tdcB gene product in

threonine degradation is well established (10); however, thephysiological significance of TdcA and TdcC is not yetunderstood. Published reports (cited in reference 22) indi-cate that most catabolic pathways, especially those involvedin the utilization of various amino acids and sugars, includea specific protein(s) for transport of metabolites into the celland that some catabolic operons, such as lac and tna, harborgenes coding for their respective permeases lacY and tnaB(4, 30). For the tdc operon, both threonine and serine arerequired for expression of the tdc genes in the synthetic H4medium (18), and thus far no E. coli gene specific forL-threonine and L-serine transport has been identified. Theseconsiderations led to the notion that one of the unassignedtdc genes may be involved in transport of the amino acids

* Corresponding author.t Present address: Department of Microbiology and Infectious

Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Can-ada.

threonine and serine. The experiments reported here showedthat the tdcC gene encodes a permease that is induced duringanaerobic incubation of E. coli in amino acid-rich medium.In analogy with other permeases, the TdcC polypeptideappears to be an integral membrane protein with severalhydrophobic domains exhibiting a polytopic feature.

MATERIALS AND METHODSChemials and enzymes. Tryptone, yeast extract, agar, and

other medium components were supplied by Difco Labora-tories (Detroit, Mich.). Antibiotics and other reagents werepurchased from Sigma Chemical Co. (St. Louis, Mo.).Restriction enzymes, DNA polymerase I (Klenow), and T4DNA ligase were bought from Bethesda Research Labora-tories, Inc. (Gaithersburg, Md.) or Boehringer MannheimBiochemicals (Indianapolis, Ind.). Sequenase (17 DNApolymerase) was supplied by United States Biochemicals(Cleveland, Ohio). Uniformly labeled L-[14C]threonine (spe-cific activity, 234 mCi/mmol) was bought from Dupont, NENResearch Products (Boston, Mass.).

Bacterial strains and growth media. E. coli MC4100 [F-araD139 A(argF-lac)U169 rpsL150 deoCI relAl rbsR ptsF25flbB5301] (29) was used in some transport experiments,whereas strain TH16 (MC4100 Atdc-216 zqi-l::TnlO) con-taining a chromosomal deletion of the tdc operon (26) wasused to measure transport by plasmid-encoded tdc genes.Strain CC202 [araDJ39 A(ara-leu)7697 AlacX74 AphoA20recAl(F42 lac-13 zzf-2::TnphoA)] (2Q) was employed forisolation of TnphoA insertions. TdcC-LacZ fusions wereassayed in strain DH5aF' [F' 480d lacZAM15 A(lacZYA-argF) U169 recAl endAl hsdR17 (rK- MK+) supE44 X- thi-lgyrA relAl] (28). LB and M9 minimal medium withoutglucose were prepared by the methods of Miller (21) andDavis et al. (11), respectively. The synthetic H4 medium wasthat of Hobert and Datta (18). The TYE medium contained2% tryptone and 1% yeast extract supplemented with saltsand pyridoxine hydrochloride (18).

Plasmids. The plasmids pEC61, pRS124, pTG122, pBal4,and pSH215 have been described previously (13, 14, 26).Plasmid pRS125 containing a frameshift mutation in tdcA

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E. COLI tdcC ENCODES A THREONINE PERMEASE

A

BpEC61

pSH215

pRS124

pRS125

pTG 122

pBad4

R A B C

X=-O-LiZiIH H

E Hp S BSc P rHp H E

E Hp Hp H EI I I I I

E S -%ScP HI I I I . IE S* B Sc P HI I I I IE S B Sc PI I I I I

E S B Sc PI I 1 1

FIG. 1. Physical organization of the tdc operon and restrictionmaps of various plasmids described in the text. Abbreviations forrestriction enzyme sites are: E, EcoRI; Hp, HpaI; S, Sall; B, BglII;Sc, Scal; P, PstI; and H, HindIll. The dashed line in pSH215indicates the deleted segment. S* in pRS125 denotes the location ofa frameshift mutation at the Sall site as described in Materials andMethods.

was constructed by digesting pRS124 with Sall, filling in therecessed ends with Klenow DNA polymerase I, and subse-quent religation. Loss of the Sall site and generation of a

PvuI site were used to screen for the frameshift mutation.The extent of tdc DNA carried by these plasmids is shown inFig. 1.

Isolation of gene fusions. The LacZ protein fusion plasmidpSH295 was constructed by ligating a 3,955-base-pairEcoRI-Scal fragment from pEC61 containing the completetdcR and tdcAB genes and the first two codons of tdcC toEcoRI-Smal-cleaved pMC1403 DNA (8). This procedurefused codon 2 of tdcC' (14) to codon 8 of lacZ. The correctconstruc,t was verified by restriction digest and DNA se-

quence analysis.For isolation of TnphoA insertions, pSH231 was con-

structed by ligating the 6,295-base-pair EcoRI fragment frompEC61 to EcoRI-cleaved pACYC184 DNA (9). In pSH231,tdcABC is transcribed in the same orientation as the catgene. A series of TnphoA insertions in pSH231 was isolatedby the method of Manoil and Beckwith (20) with strainCC202. Some of the fusion plasmids (pSH231::TnphoA)were stabilized by deleting the transposase and the npt gene

as described by Gott and Boos (15), yielding the pCX seriesof plasmids. The sequences of the fusion junctions were

determined by using a TnphoA-specific primer (15) aftersubcloning the PstI-EcoRI fragments (from nucleotide 4511in tdcC' to the EcoRI site in phoA) from the pCX plasmidsinto M13mpl9.Enzyme assay. Threonine dehydratase activity of cells

incubated anaerobically in TYE or H4 was assayed colori-metrically with toluene-treated cells as described previously(18). P-Galactosidase and alkaline phosphatase activities insodium dodecyl sulfate-chloroform-permeabilized cells wereassayed as described by Miller (21) and Gott and Boos (15),respectively. Alkaline phosphatase and NADH oxidase ac-

tivities of membrane and soluble fractions (see below) were

measured by the methods of Schweizer et al. (25) andOsbom et al. (23), respectively. Specific activities of threo-nine dehydratase and NADH oxidase are expressed as

micromoles of products formed per minute per milligram ofprotein; P-galactosidase and alkaline phosphatase activities

are expressed as milliunits per minute per milligram ofprotein.

Cell fractionation. For preparations of membrane andsoluble fractions, cells suspended in 10 mM Tris hydrochlo-ride buffer (pH 7.5) were disrupted by passage through aFrench pressure cell (16,000 lb/in2). To the extract wasadded phenylmethylsulfonyl fluoride at a concentration of 20,ug/ml, and unbroken cells were removed by centrifugation at10,000 x g for 10 min. The supernatant fluid (crude extract)was subjected to further centrifugation at 110,000 x g for 2h. The supernatant fluid (soluble fraction) was carefullywithdrawn, and the pellet was resuspended in 10 mM Trishydrochloride buffer (pH 7.5) containing 20 ,ug of phenyl-methylsulfonyl fluoride per ml (membrane fraction). In sep-arate experiments, periplasmic proteins were isolated by thecold osmotic shock procedure of Higgins and Hardie (17).During cell fractionation, all maniipulations were done at4°C. Protein concentration was determined by the method ofBradford (7), with bovine serum albumin as the standard.

Transport assay. Cells grown aerobically for 10 to 20 h at37°C in TYE medium were harvested, washed in 100 mMpotassium phosphate buffer (pH 7.2), and suspended in freshTYE or H4 medium. To induce the tdc operon, we incubatedthe cells anaerobically (in still culture) for 4 to 5 h at 37°C.After incubation, cells were collected, washed twice in M9minimal medium without any carbon source, and resus-pended in the same medium containing 50 ,g of chloram-phenicol per ml at a cell density of 2 x 108 cells per ml. Themixture was further incubated for 30 to 60 min at 37°Cwithout shaking to lower the endogenous pool of amino acidsbefore transport assay. A 3-ml portion of cell suspension wasmixed with 0.1 mM L-[14C]threonine to achieve a finalconcentration of 10 ,uM L-threonine. Samples of 0.7 ml of themixture (containing approximately 100,000 cpm) were with-drawn at 30-s intervals for transport assay at 25°C by filteringthrough Millipore filters (2). The amount of radioactivityretained by the washed, dried filters was measured byscintillation counting in ScintiVerse cocktail mixture (FisherScientific Co., Pittsburgh, Pa.). Background counts retainedby the filters without cells were <0.5% of total counts andwere subtracted before calculating the amount of threonineuptake. The data are expressed as nanomoles Of L-threonineuptake per minute per milligram of protein from averages ofduplicate or triplicate assays; individual values were within10% of average.

RESULTS

Threonine uptake in tdc strains. In E. coli K-12 strains, atleast two separate transport systems for L-threonine areknown: the serine-threonine system presumably associatedwith the cytoplasmic membrane (19, 31), and one of the twoleucine-isoleucine-valine systems (LIV-1) which transportsseveral amino acids including alanine, serine, and threoninein addition to the branched-chain amino acids (see review byAntonucci and Oxender [3]). Recently, a leucine-inducedtransport system highly specific for L-serine has been iden-tified which cotransports with H' (16). In all cases citedabove, cells were grown aerobically in minimal medium withglucose or lactate as the carbon source, occasionally supple-mented with amino acids (16). The results presented belowindicate that E. coli cells exhibit a novel threonine transportactivity that requires the participation of a gene product ofthe anaerobically expressed tdc operon.When incubated anaerobically in H4 medium containing

the L-isomers of threonine, serine, valine, and isoleucine

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4290 SUMANTRAN ET AL.

TIME (seconds)FIG. 2. Threonine uptake by MC4100 (tdc+) and TH16 (Atdc)

strains. Before the transport assay, cells were incubated anaerobi-cally in complete H4 medium (18) (O, 0) or in H4 medium withoutfumarate (A) or without cyclic AMP (A). For details of cell growthand transport measurements, see Materials and Methods. The dataare expressed as nanomoles of L-ihreonine uptake per milligram ofprotein. Symbols: 0, A, A, MC4100 (tdc'); X, TH16 (Atdc).

plus cyclic AMP and fumarate (18), strain MC4100 showedhigh uptake of L-threonine in a time-dependent manner (Fig.2). Omission of cyclic AMP or fumarate from the H4 mediumresulted in a low level of uptake activity. In this experiment,threonine dehydratase activity in MC4100 in H4 meditim was14 U/mg of protein; no enzyme activity was detected in thecyclic AMP- or fumarate-deficient H4 medium. Previousresults showed that both cyclic AMP and fumarate areabsolutely required. for tdc operon expression in H4 medium(13, 14, 18). Unlike MC4100, strain TH16, an isogenicderivative of MC4100 harboring a chromosomal deletion ofthe tdc genes and lacking dehydratase activity (26), exhibiteda basal leVel of L-threonine uptake in H4 (Fig. 2). These datasuggest that a tdc gene product expressed anaerobically isinvolved in L-threonine transport. It is likely that the basallevel of L-threonine uptake seen in the absence of the tdcgenes or in the wild-type strain under noninducing condi-tions reflects other threonine transport systems such asLIV-1 (3).To determine whether the cloned tdc genes would restore

a high level of transport of L-threonine in TH16 (Atdc), wemeasured threonine uptake rates in TH16 transformed withthe plasmids pEC61 (tdc+) and pSH215 (Atdc) (Fig. 3). Ahigh level of L-threonine uptake was observed inTH16(pEC61) only when it was incubated anaerobically instill culture in TYE medium but not when it was incubatedaerobically with shaking. Aerobic incubation of E. coli inTYE medium produces very little, if any, biodegradativethreonine dehydratase activity (18). In comparison, strainTH16 containing the tdc deletion plasmid pSH215 showed alow level of L-threonine uptake regardless of aerobic oranaerobic incubation conditions. Thus, the cloned tdc DNAeffectively complements the chromosomal tdc mutation inTH16 in terms of L-threonine transport activity during an-aerobiosis.Requirement for tdcC for threoIne trasport. Because

tdcB is known to be the structural gene for biodegradativethteotine dehydratase (10, 13), the foregoing experimentssuggest that the tdcA and/or tdcC gene products are likely tobe involved in amino acid transport. Figure 4 displays theresults of L-threonine uptake rates with strain TH16 (Atdc)

TIME (cons)FIG. 3. Restoration of threonm transport in TH16 (Atdc) by

cloned tdc DNA. Strain TH16 (Atdc) was trasformed with pEC61(tdWe) or pSH215 (Atdc), and the transformants were incubated inTYE medium anaerobically in still culure (0, A) or aerobically withshaking (*, A) before transport assays. For details of cell growthand transport measurements, see Materials and Methods. The dataare expressed as nanomoles of L-thronnie uptake per milligam ofprotein. Symbols: 0, 0, TH16(pEC61); A, A, TH16(pSH215).

transformed with mutant plasmids containing a functionaltdcA or tdcC; control plasmids included were pSH215 (Atdc)and pRS124 (tdc+) (Fig. 1). It is clear that the transformantswith a truncaited tdcC gene, as in TH16(pBal4) and TH16(pTG122), with or without tdcR, respectively, showed lowtransport activity similar to that observed in TH16(pSH215),containing deletions of both the chromosomal and plasmid-borne tdc operons. The involvement of the tdcC geneproduct in threonine transport was also confirmed in sepa-rate experiments with tdc-lacZ fusion pasmids. Neither thetdcA'-'1acZ nor tdcB'-'1acZ fusion (28), both lacking tdcC,showed a higher level of threonine uptake than that seen inthe Lac- TH16 background during anaerobic incubation inTYE, whereas both fusions expressed [-galactosidase activ-ity (data not shown).

TIM (tconns)FIG. 4. Threonine transport by various mutant plasmids. Strain

TH16 (Atdc) was transformed separately with various plasmids asindicated below, and the transformants were intcibated anacrobi-cally in TYE medium before transport assays. For dets of cellgrowth and transport measurements, see Materials and Methods.The data are expressed as nanomoles of L-threonine uptake permilligram of protein. Symbols: 0, TH16(pRS124); 0, TH16(pRS125); A, TH16(pBal4); A, TH16(pTG122); O, TH16(pSH21S).For the extent of the tdc DNA carried by these plasmids, see Fig. 1.

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E. COLI tdcC ENCODES A THREONINE PERMEASE 4291

TABLE 1. Specificity of threonine transport system

Amino acids added Relative threonine(0.5 mM) uptakea

None..................................... 1.0L-Alanine ..................................... 0.81L-Valine ..................................... 0.96L-Isoleucine ..................................... 0.96L-Leucine ..................................... 0.60L-Homoserine ..................................... 0.42L-Serinc ..................................... 0.23

a Average values determined in two separate experiments. Threonineuptake by TH16(pEC61) in the absence of amino acid was 3.46 nmol min-I mgof protein-'. As a control, the relative threonine uptake by TH16(pSH215)was 0.20.

Anaerobic incubation of strain TH16 (Atdc) transformedwith pRS125, which contains a frameshift mutation in tdcA(Fig. 1), showed an intermediate level of L-threonine uptakecompared with that observed with tdcC+ and tdcC plasmids(Fig. 4). Because the same vector was used in all plasmidconstructions, it is unlikely that the differences in threonineuptake were due to plasmid copy number effects. This resultsuggests that tdcA has some indirect effect on tdcC-mediatedtransport of threonine. This notion is consistent with therecent findings in this laboratory (S. R. Sadda and P. Datta,unpublished data) that a mutation in tdcA which somehowinterferes with the synthesis of the inducing metabolite(s)prevents optimum expression of the tdc operon. In addition,transformants harboring the cloned tdcA gene alone exhib-ited the same low basal levels of threonine transport as seenin the host TH16 (data not shown).

Characterization of transport system. The specificity ofL-threonine transport by the anaerobically induced tdcCgene product was tested by including a 50-fold molar excessof unlabeled amino acids in the transport assay mixtures inthe presence of L-[14C]threonine (Table 1). The amino acidsL-alanine, L-valine, and L-isoleucine did not affect L-threo-nine uptake significantly, whereas L-leucine inhibited L-threonine uptake by about 50%. L-Homoserine, a metabolicintermediate structurally related to threonine, competedeffectively with L-threonine similar to that reported byTempleton and Savageau (31). Addition of L-serine, how-ever, resulted in complete loss of threonine uptake as judgedfrom that seen with TH16(pSH215). Thus, the tdcC geneproduct appears to mediate transport of both L-threonineand L-serine in E. coli cells; the same transport system maybe partially shared by L-leucine, which is also transported by

the LIV-1 and LIV-2 systems (3). L-Threonine uptake byTdcC showed a typical saturation kinetics as a function ofL-threonine concentration, and the Km for L-threonine intransport assays in TH16(pEC61) was approximately 6 p.M.To test the possibility that uptake of L-threonine by TdcC

is an active process, we performed transport assays in thepresence of several energy-coupling inhibitors. At 10 and 20,uM, the proton conductor carbonyl cyanide m-chlorophen-ylhydrazone inhibited the transport process by 83 and 100%,respectively. The respiratory chain inhibitor KCN also in-hibited L-threonine uptake by 50 and 70% at concentrationsof 5 and 10 mM, respectively. The Na+-H+ antiportermonensin, on the other hand, had no effect on L-threonineuptake at a concentration of 50 F.M in the presence of 10 mMNaCl (data not shown), indicating that transport of L-threonine does not appear to be coupled with Na+ cotrans-port. These data taken together suggest that a proton gradi-ent is most likely the driving force for active transport ofL-threonine.Membrane origin of TdcC. Goss et al. (14) reported that

the TdcC polypeptide is highly hydrophobic, with 46% of allamino acid residues being hydrophobic in nature. In addi-tion, the hydropathy plot of the amino acid sequence re-vealed alternating hydrophilic and hydrophobic segments, afeature characteristic of integral membrane proteins. Atwo-dimensional topological model of TdcC, as predictedfrom the hydropathy plot (14), is displayed in Fig. 5. The 12membrane-spanning domains of the polypeptide show re-markable similarity with several other polytopic integralmembrane proteins such as MalF (6), SecY (1), and GlpT(15). In accordance with the prediction of von Heijne (33),most of the positively charged residues in TdcC appear in thecytoplasmic loops, including both the N and C termini. Toexperimentally test this topological model, we used thegenetic approach of phoA fusions to tdcC. This method hasbeen used to characterize transmembrane topology and toidentify hydrophobic domains which function as internalsignal sequences (6, 15, 20, 24). The procedure is based onthe observation that when the catalytic moiety of alkalinephosphatase, a periplasmic protein that must be locatedextracytoplasmically to be active (5), is fused to a proteinfound in the cytoplasm, it exhibits little or no activity. On theother hand, the phoA-protein fusions appearing on theperiplasmic side of the membrane exhibit high phosphataseactivity. No exceptions have yet been documented thatobviate the prediction.The 6.3-kilobase insert in pSH231 (same as pEC61 but

FIG. 5. Two-dimensional topological model of TdcC polypeptide as predicted from amino acid composition and hydropathy plot (14).Numbers next to horizontal lines refer to amino acid residues. Numbers I through XII represent transmembrane segments. Charged aminoacids are shown with + or -. The position of alkaline phosphatase fusions in pCX1 through pCX4 at Asp-386 of TdcC' is indicated by anarrow.

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4292 SUMANTRAN ET AL.

TdbC' 'PhoA

A ATC CT 1AC CC AC TaCI L D P D S

386 L9 Z

TdeC! IWWZ

B ATG AGT OAT CCC

S G D P

1 2

FIG. 6. Sequences of TdcC'-PhoA and TdcC'-LacZ fusion junc-tions. Amino acid residues are indicated by one-letter codes. Thenumbers designate the residue numbers of TdcC.

cloned into pACYC184) encodes tdcR, tdcABC, and threeother open reading frames (14, 27, 28), of which only TdcCexhibits characteristics of a membrane protein (14). Restric-tion analysis of eight TnphoA insertions in pSH231 withmoderate to high alkaline phosphatase activity indicated thatall these insertions were located in the vicinity of the singlePstI site within tdcC (Fig. 1). Four of these fusions, pCX1through pCX4, were chosen. for further studies. Alkalinephosphatase activities of these fusions ranged from 18 to 20mU/mg of protein during aerobic (noninducing) growth inTYE medium (A6. = 0.8) and from 290 to 300 mU/mg ofprotein during anaerobic incubation in TYE medium (7.5 h instill culture), which allows tdc operon expression. DNAsequence analyses of the fusion junctions in the plasmidsindicated that they were identical. In all four cases, aminoacid residue Asp-386 of TdcC' was fused to alkaline phos-phatase (Fig. 6A); this residue appears to represent a hot-spot target site for TnphoA transposition in tdcC similar toseveral hot spots found in the gipT gene of E. coli (15). Anexamination of the model presented in Fig. 5 shows that thefusions are located in the last periplasmic loop of the TdcCpolypeptide.

Surprisingly, the subcellular distribution of alkaline phos-phatase encoded by pCX1 showed that 87% of total enzymeactivity was in the soluble fraction, with the remaining 13%of the activity found in the membrane fraction (Table 2).Furthermore, cold osmotic shock treatment of DHSaF'(pCX1) cells released aLkaline phosphatase enzyme in theperiplasmic fluid. A companson of protein patterns in theshock fluids of cells grown under aerobic (uninduced) andanaerobic (induced) conditions revealed the presence of a

TABLE 2. Subcellular distribution of TdcC'-PhoA activity

Alkaline phosphatase NADH oxidaseFraction" ,umoV/min % Total lmol/min % Total

per mg activity per mg activity

Membrane 0.023 13 0.368 99.2Soluble 0.155 87 0.003 0.8Shock fluid 1.143 NDC ND

a Strain DH5aF' harboring pCX1 was grown anaerobically overnight inTYE medium containing 10 "g of tetracycline per ml. Subceliular fractionswere prepared as described in Materials and Methods, and enzymatic activ-ities were measured spectrophotometrically.

b This value was obtained by comparing the activities measured in wholecells, in whole shocked cells, and in shock fluid.

' ND, Not determined.

unique protein of M, 50,000 in the latter (data not shown).The size of this protein was considerably smaller than thecalculated Mr of 92,000 for the TdcC'-PhoA fusion proteinbut was slightly larger than the size of mature alkalinephosphatase (Mr 47,000). It would appear then that theoriginally synthesized fusion protein was proteolyticallyprocessed into a smaller, active TdcC'-aLkaline phosphatasefusion protein after its translocation across the membrane.In any case, the data summarized above suggest that theTdcC polypeptide is most likely an integral membrane pro-tein exhibiting a polytopic feature.

E*presuion of tdcC. The notion of tdcC being part of thetdcABC operon was derived from the DNA sequence dataand minicell analysis of gene products of insertion mutants(14, 27). However, until now no direct genetic and functionalevidence existed to support this conclusion. The experi-ments reported above clearly show that specific mediumcomposition and growth conditions were crucial for coordi-nated synthesis of the tdcB and tdcC gene products asmeasured, respectively, by dehydrata activity and threo-nine transport. Under noninducing conditions, lack of TdcBactivity from tdc+ DNA accompanied lack of transportactivity. This finding was extended by monitoring 0-galac-tosidase activity encoded by a tdcC'-'lacZ fusion plasmid, inwhich codon 2 of tdcC' was fused with codon 8 of 'lacZ (Fig.6B). The strain DH5aF' (Alac tdc+) transformed with theplasmid pSH295 (tdcA+B+C'-'lacZ) had, per mgram ofprotein, <0.01 U of dehydratase activity and <10 mU of,-galactosidase activity during (noninducing) aerobic growthin TYE medium (A.. = 0.7). In comparison, the dehy-dratase and 3-galactosidase activities during anaerobic incu-bation in TYE medium (8.5 h) showed, respectively, 11.0U/mg of protein and 200 mU/mg of protein (after subtractionof 0.64 U of dehydratase activity per mg of protein owing tothe chromosomally encoded tdcB gene). Thus, TdcC'-'LacZ-specified 3-galactosidase activity is coinduced withthreonine dehydratase in TYE medium under anaerobic(inducing) culture conditions. These data corroborate theprevious findings that tdcC is indeed a member of thepolycistronic tdc operon.

DISCUSSIONIdentification of the function of a gene product is an

essential first step to study its physiological role in vivo.Previous studies showed that the tdcC gene of the tdcABCoperon ofE. coli encodes a large hydrophobic polypeptide of431 residues (14). A search of the 1989 Protein SequenceDatabase (Protein Identification Resource of the NationalBiomedical Research Foundation, Washington, D.C.) failedto detect any significant homology of TdcC with any otherknown proteins. The experiments summanzed here showthat E. coli cells incubated anaerobically in amino acid-richmedia induced a novel L-threonin transport system thatrequires a functional TdcC protein. No inducible L-threonineuptake was seen in the absence of tdc operon expression.The transport system showed a Km of 6 uM for L-threonine,was Na+ independent, and requied a source of metabolicenergy for amino acid uptake. Competition experimentsrevealed that both L-threonine and L-serine shared the sametransport system. The following findings suggest that theanaerobically expressed TdcC-mediated L-threonine trans-port is clearly distinct from other threonine-serine uptakesystems. The genes for the LIV-1 system have been clonedand sequenced and mapped on the E. coli chromosome atminute 76 (3), whereas the tdc operon is located at minute 68

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E. COLI tdcC ENCODES A THREONINE PERMEASE 4293

(26); the leucine-induced highly specific L-serine transportsystem (16) is not affected by preincubation of the cells withexcess amino acids including L-threonine; and the threonine-serine transport systems reported by Lombardi and Kaback(19) and by Templeton and Savageau (31) were found in cellsgrown aerobically in minimal medium with glucose or lactateas the carbon source. It is assumed that the basal level ofthreonine uptake observed in the absence of a functionaltdcC gene is due to one of these amino acid transportsystems. It should be mentioned in this context that Eganand Phillips (12) detected L-threonine uptake by E. coliCrookes strain grown anaerobically in glycerol-fumaratemedium in the presence or absence of 19 common aminoacids but excluding L-threonine; however, the transportsystem was not further characterized. Thus, the genetic andbiochemical evidence reported here identifies for the firsttime an E. coli gene that encodes a permease specific forL-threonine-L-serine transport.A topological model predicted from the amino acid se-

quence and hydropathy plot of the TdcC polypeptide re-vealed 12 membrane-spanning domains with a large, innercytoplasmic loop. A preliminary support for this model wasobtained from the PhoA fusion study. The location of ahot-spot target site for PhoA insertion in the last periplasmicloop suggests that the hydrophobic segment XI (Fig. 5)functions as a putative internal signal sequence. A detailedanalysis by a combined phoA and lacZ fusion approach asdescribed by Gott and Boos (15) should clarify the validity ofthe model. Interestingly, the overall structural features ofTdcC exhibit a striking similarity with the sn-glycerol-3-phosphate permease, GlpT (15). Several other bacterialpermeases such as LacY, MelB, and AraE also show similartopology, with 12 transmembrane segments, and it is spec-ulated (15) that this common architectural feature is essentialfor one-component membrane transport systems. By anal-ogy with these permeases, TdcC appears to be an integralmembrane protein with threonine permease activity. Thistype of transport system does not appear to require a bindingprotein.

In physiological terms, it is not surprising that a memberof the tdc operon encodes a threonine-serine permease. Asmentioned above, the minimal conditions required for anaer-obic induction of tdc genes are four amino acids, threonine,serine, valine, and isoleucine, plus cyclic AMP and fuma-rate. In addition, L-threonine and L-serine are the substratesfor the threonine dehydratase coded by the tdcB gene. Thistype of genetic organization, in which a single transcriptionalunit contains genes coding for both a permease and anenzyme needed for catabolism of a specific metabolite, as inlac and tna operons, provides a biochemical mechanism forcoordinate expression of the gene products for efficientregulation of cellular metabolism.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantGM21436 from the National Institutes of Health.We thank Dale L. Oxender for critically reviewing the manu-

script.

LITERATURE CITED1. Akiyama, Y., and K. Ito. 1987. Topology analysis of the SecY

protein, an integral membrane protein involved in protein ex-port. EMBO J. 6:3465-3470.

2. Anderson, J. J., S. C. Quay, and D. L. Oxender. 1976. Mappingof two loci affecting the regulation of branched-chain amino acidtransport in Escherichia coli K-12. J. Bacteriol. 126:80-90.

3. Antonucci, T. K., and D. L. Oxender. 1986. The molecularbiology of amino-acid transport in bacteria. Adv. Microb.Physiol. 27:145-180.

4. Beckwith, J. 1987. The lactose operon, p. 1444 1452. In F. C.Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M.Schaechter, and H. E. Umbanger (ed.), Escherichia coli andSalmonella typhimurium: cellular and molecular biology, vol. 2.American Society for Microbiology, Washington, D.C.

5. Boyd, D., C. Guan, S. Willard, W. Wright, K. Strauch, and J.Beckwith. 1986. Enzymatic activity of alkaline phosphataseprecursor depends on its cellular location, p. 89-93. In A.Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright, and E.Yagil (ed.), Phosphate metabolism and cellular regulation inmicroorganisms. American Society for Microbiology, Washing-ton, D.C.

6. Boyd, D., C. Manoil, and J. Beckwith. 1987. Determinants ofmembrane protein topology. Proc. Natl. Acad. Sci. USA 84:8525-8529.

7. Bradford, 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.

8. Casadaban, M. J., J. Chou, and S. N. Cohen. 1980. In vitro genefusions that join an enzymatically active P-galactosidase seg-ment to amino-terminal fragments of exogenous proteins: Esch-erichia coli plasmid vectors for the detection and cloning oftranslation initiation signals. J. Bacteriol. 143:971-980.

9. Chang, A. C. Y., and S. N. Cohen. 1978. Construction andcharacterization of amplifiable multicopy DNA cloning vehiclesderived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141-1156.

10. Datta, P., T. J. Goss, J. R. Omnaas, and R. V. Patil. 1987.Covalent structure of biodegradative threonine dehydratase ofEscherichia coli: homology with other dehydratases. Proc.Natl. Acad. Sci. USA 84:393-397.

11. Davis, R. W., D. Botstein, and J. Roth (ed.). 1980. Advancedbacterial genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

12. Egan, R. M., and A. T. Phillips. 1977. Requirements for induc-tion of the biodegradative threonine dehydratase in Escherichiacoli. J. Bacteriol. 132:370-376.

13. Goss, T. J., and P. Datta. 1985. Molecular cloning and expres-sion of the biodegradative threonine dehydratase gene (tdc) ofEscherichia coli K-12. Mol. Gen. Genet. 201:308-314.

14. Goss, T. J., H. P. Schweizer, and P. Datta. 1988. Molecularcharacterization of the tdc operon of Escherichia coli K-12. J.Bacteriol. 170:5352-5359.

15. Gott, P., and W. Boos. 1988. The transmembrane topology ofthe sn-glycerol-3-phosphate permease of Escherichia coli anal-ysed by phoA and lacZ protein fusions. Mol. Microbiol. 2:6554663.

16. Hama, H., T. Shimamoto, M. Tsuda, and T. Tsuchiya. 1988.Characterization of a novel L-serine transport system in Esch-erichia coli. J. Bacteriol. 170:2236-2239.

17. Higgins, C. F., and M. M. Hardie. 1983. Periplasmic proteinsassociated with oligopeptide permeases of Escherichia coli andSalmonella typhimurium. J. Bacteriol. 155:1434-1438.

18. Hobert, E. H., and P. Datta. 1983. Synthesis of biodegradativethreonine dehydratase of Escherichia coli: role of amino acids,electron acceptors, and certain intermediary metabolites. J.Bacteriol. 155:586-592.

19. Lombardi, F. J., and H. R. Kaback. 1972. Mechanisms of activetransport in isolated bacterial membrane vesicles. VIII. Thetransport of amino acids by membranes prepared from Esche-richia coli. J. Biol. Chem. 247:7844-7857.

20. Manoil, C., and J. Beckwith. 1985. TnphoA, a transposon probefor protein export signals. Proc. Natl. Acad. Sci. USA 82:8129-8133.

21. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

22. Neidhardt, F. C., J. L. Ingraham, K. B. Low, B. Magasanik, M.Schaechter, and H. E. Umbarger (ed.). 1987. Escherichia coliand Salmonella typhimurium: cellular and molecular biology.American Society for Microbiology, Washington, D.C.

VOL. 172, 1990

on February 25, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

4294 SUMANTRAN ET AL.

23. Osborn, M. J., J. E. Gander, E. Parisi, and J. Carson. 1972.Mechanism of assembly of the outer membrane of Salmonellatyphimurium. J. Biol. Chem. 247:3962-3972.

24. Saier, M. H., P. K. Werner, and M. MflUer. 1989. Insertion ofproteins into bacterial membranes: mechanism, characteristics,and comparisons with the eukaryotic process. Microbiol. Rev.53:333-366.

25. Schweizer, H., M. Arpst, and W. Boos. 1982. Characteristics ofa binding protein-dependent transport system for sn-glycerol-3-phosphate in Escherichia coli that is part of the pho regulon.J. Bacteriol. 150:1154-1163.

26. Schwelzer, H. P., and P. Datta. 1988. Genetic analysis of thetdcABC operon ofEscherichia coli. J. Bacteriol. 170:5360-5363.

27. Schwelzer, H. P., and P. Datta. 1989. The complete nucleotidesequence of the tdc region of Escherichia coli. Nucleic AcidsRes. 17:3994-3998.

28. Schweizer, H. P., and P. Datta. 1989. Identification and DNA

sequence of tdcR, a positive regulatory gene ofthe tdc operon ofEscherichia coli. Mol. Gen. Genet. 218:516-522.

29. Silhavy, T. J., M. L. Berma, and L. W. Enquist. 1984.Experiments with gene fusions. Cold Spring Harbor Labora-tory, Cold Spring Harbor, N.Y.

30. Stewart, V., and C. Yanofsky. 1985. Evidence for transcriptionantitermination control of tryptophanase operon expression inEscherichia coli K-12. J. Bacteriol. 164:731-740.

31. Templeton, B. A., and M. A. Savageau. 1974. Transport ofbiosynthetic intermediates: homoserine and threonine uptake inEscherichia coli. J. Bacteriol. 177:1002-1009.

32. Umbarger, H. E. 1978. Amino acid biosynthesis and its regula-tion. Annu. Rev. Biochem. 47:533-606.

33. von Heijne, G. 1986. The distribution of positively chargedresidues in bacterial inner membrane proteins correlates withthe trans-membrane topology. EMBO J. 5:3021-3027.

J. BACTERIOL.

on February 25, 2021 by guest

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Dow

nloaded from