JOURNAL OF BACTERIOLOGY, May 1982, p. 722-7290021-9193/82/050722-08$02.00/0

Vol. 150, No. 2

Role of the Catabolite Activator Protein in the MaltoseRegulon of Escherichia coli

CHRISTINE CHAPONUnite de GfnEtique Mo61culaire, Institut Pasteur, 75724 Paris Cedex IS, France

Received 5 August 1981/Accepted 10 December 1981

The maltose regulon consists of three operons controlled by a positiveregulatory gene, malT. Deletions of the gene crp were introduced into strainswhich carried a malT-lacZ hybrid gene. From the observed reduction in ,B-galactosidase activity it was concluded that the expression of malT-lacZ, andtherefore of malT, is controlled by the catabolite activator protein (CAP), theproduct of the gene crp. Mutations were obtained which allowed a malT-lacZhybrid gene to be expressed at a high level even in the absence of CAP. Thesemutations were shown to be located in or close to the promoter of the malT geneand were called malTp. The malTp mutations were transferred in the cis positionto a wild-type malT gene. In the resulting strains, the expression of two of themaltose operons, malEFG and malK-lamB, still required the action of CAP,whereas that of the third operon, malPQ, was CAP independent. Therefore, inwild-type cells, CAP appears to control malPQ expression mainly, if not solely,by regulating the concentration of MalT protein in the cell. On the other hand, itcontrols the other two operons more stringently, both by regulating malTexpression and by a more direct action, probably exerted on the promoters ofthese operons.

The maltose regulon of Escherichia coli con-sists of three operons, malPQ, malK-lamB, andmalEFG, controlled by a positive regulatorygene, malT (Fig. 1). The full expression of thisregulon, like that of several other genes involvedin the catabolism of sugars (2, 25), requires theaction of cyclic AMP (cAMP) and of its recep-tor, the catabolite activator protein (CAP). In-deed, deletions of the gene crp, which codes forCAP, or of the gene cya, which codes foradenylate cyclase, result in a Mal- phenotype(8, 16). However, very little is known regardingthe site(s) of action of CAP and cAMP in themaltose regulon. The recent finding that malTexpression was stimulated by cAMP (2) suggest-ed that the Mal- phenotype of crp or cya strainscould result from a deficit of MalT protein. Thispossibility was examined in the present work.Using malT-lacZ fusion strains, we confirmedthat malT expression is controlled by CAP. Wethen obtained mutations, probably located in thepromoter region of malT, which led to the syn-thesis of large amounts of MalT protein in theabsence of CAP. In such mutants, CAP was stillrequired for maximum expression of the malK-lamB and malEFG operons. Therefore, the ac-tion of CAP in the maltose regulon cannot besolely explained by its action on malT expres-sion.

MATERIALS AND METHODSStrains and media. All strains were derived from E.

coli K-12 and are described in Table 1. Standard phagestrains (X Vho, 080 vir, A cI h80, 480 psuIlI, 4)80dmaLA2, P1 vir, Mu) were from our laboratory collec-tion (11). Phage A pmalA3 and a derivative of Ap4>(malT-lacZ)S42-i(Hyb) carrying the amber muta-tion malT250 in the malT portion of the hybrid genewere recently described (3, 19). Phage A dcrp+ hadbeen isolated by N. Fiil (personal communication) andwas given to us by B. Gicquel-Sanzey. Completemedium (ML) and synthetic medium (M63) were pre-viously described (10).

Assays. The enzymes ,-galactosidase (,B-D-galacto-side galactohydrolase; EC 3.2.1.23) and amylomalt-ase (1,4-a-D-glucan:1,4-a-glycosyltransferase; EC2.4.1.25) were assayed according to Miller (14) andSchwartz (23).The LamB protein is the receptor for phage lambda

(20). It was assayed as before (24) by its ability toinactivate A Vho in vitro. However, the extractionprocedure was modified as described below becausethe previously used technique had been recently foundto be inefficient (7). Exponentially growing bacteriawere harvested, suspended at an optical density at 600nm of 3.0 in 10 mM Tris-hydrochloride (pH 7.5)-5 mMEDTA-3% Triton X-100, and incubated for 1 h at37°C. The resulting extract was then used as before toperform the assay. By using this technique and thepreviously established correlation between the rateconstant for phage inactivation and the concentrationof receptor (24), the number of active receptor mole-

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ROLE OF CAP IN E. COLI MALTOSE REGULON 723

.I

na/Q ma/P % malT

=1I~

AmylomakasePi

Maltose

maiG ma/F malE marr/K IamB

- i I d I l lI I IIIMaleto Receptorbinding for phageprotein lambda

FIG. 1. The maltose regulon in E. coli. The direction of transcription of the various genes is shown byinterrupted arrows. The malPQ operon codes for the enzymes specific to the metabolism of maltose andmaltodextrins (9). The proteins coded by the genes located in the malEFG and malK-lamB operons are allinvolved in the transport of maltose and maltodextrins across the bacterial envelope (18, 26). malTis the positiveregulatory gene (2, 9). According to the current model, the MalT product exists in an equilibrium between twoconformations: Pl, which is inactive, and P2, which promotes the transcription of the three mal operons.Maltose would induce the system by binding preferentially to the P2 conformation, thereby displacing theequilibrium toward the active form of the MalT product.

cules per bacterium was found to be about 5 x 104 infully induced wild-type cells. This value is about fivetimes higher than that previously estimated and is inmuch better agreement with the amount of LamBpolypeptide present in the cells, i.e., about 10i copies(1). The new extraction technique gave the samerelative values as before when different strains werecompared or when the same strains were grown underdifferent conditions.

Total protein was assayed according to the tech-nique ofLowry et al., using bovine serum albumin as astandard (13).

Isolation of the maffp mutants. Eight different clonesof strains pop3697, pop3698, pop3699, and pop3700were grown in complete medium (ML) at 37°C andplated at various dilutions (10w to 1010 bacteria perplate) on minimal lactose agar. After 48 h a firstgeneration of mutants appeared. However, these mu-tants had not only acquired the ability to utilizelactose, but also grew faster in complete medium andgrew on glycerol synthetic medium; they had other-wise retained the inability to metabolize xylose, man-

Strain

pop1239pop1235pop2170pop2171pop3pop3661pop3653pop3650pop3692pop3693pop3697pop3698pop3699pop3700pop3325pop3657

nitol, and melibiose, a characteristic of crp mutants(22). Since similar glycerol-positive mutants also ap-peared in Acrp strains which did not carry a malT-lacZhybrid gene, we did not study them any further. Asecond generation of Lac' colonies appeared muchlater, after 10 to 20 days of incubation. These werereisolated on minimal glucose agar containing 5-bro-mo-4-chloro-3-indolyl-p-D-galactoside, an indicatordye which turns blue when the cells contain 3-galacto-sidasis activity (14). These strains were Lac' butotherwise displayed the same growth pattern as theparental Acrp strains.

Genetic mapping of the maffp mutations. The tech-nique used for genetic mapping required the introduc-tion of a mutation inactivating a malT gene in the cisposition to the malTp mutation. To do this, we lysoge-nized the malTp strains with phage Mu and selectedthe Mu lysogens which had become Mal- and resistantto lambda. A rapid mapping technique, using adequatetransducing phages (19), allowed us to select strainsbearing a Mu insertion in the third or fourth deletioninterval in malT. The malTp mutations were mapped

TABLE 1. E. coli K-12 strains usedGenotype

HfrG6 Acrp-39

HfrG6 Acrp-96HfrG6 AmIAS10HfrG6 AmaA11F- araDl39 AlacUl69 rpsL relA thipop3 AmaL4S06pop3 aroB glpDpop3 aroB malT131pop3 aroB (maIT-lacZ)S44-5(Hyb)pop3 aroB (malT-lacZ)542-l(Hyb)pop3 (malT-lacZ)544-5(Hyb) Acrp-39pop3 (malT-lacZ)544-5(Hyb) Acrp-96pop3 4(malT-lacZ)S42-l(Hyb) Acrp-39pop3 (malT-lacZ)542-l(Hyb) Acrp-96pop3 malTlCpop3 malT131

Origin

J. Beckwith and B. Gicquel-Sanzey (21)J. Beckwith and B. Gicquel-Sanzey (21)0. Raibaud0. RaibaudStrain MC4100 from M. Casadaban (2)0. Raibaud (19)M. DEbarbouill6 (3)Transduction of pop3653 by P1 grown on pop3657M. D6barbouilliM. Debarbouill6Transduction of pop3692 by P1 grown on pop1239Transduction of pop3692 by PI grown on pop1235Transduction of pop3693 by P1 grown on popl239Transduction of pop3693 by P1 grown on pop1235M. D6barbouill6M. D6barbouillf (3)

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724 CHAPON

controlregion Mu malT

I *I , - I I

1 2

£511 malT

controlregion

a -IMu malT

A 510 malT

FIG. 2. Mapping of the mutations. (a) Genetic structure of the F- strain. The 11 intervals in malT werepreviously defined by the endpoints of deletions or of DNA segments carried by transducing phages (19;M. Debarbouille, P. Cossart, and 0. Raibaud, Mol. Gen. Genet., in press). The F- strain contains one of themutations studied (0) and a Mu insertion (U). (b) Genetic structure of the Hfr strains. One of them (pop2171carries deletion AmaLAS l, which ends 146 base pairs before the initiation codon in malT (Debarbouill6 et al., inpress). The other, pop2170, carries AmaIA510, whose extremities are no more than 100 base pairs after theinitiation codon in malT. The deletions are shown as open bars.

by crossing these malTp malT::Mu F- strains with Hfrstrains carrying the deletion AmalA510 or Amal45l1.Mal' His' recombinants were selected and screenedfor constitutive amylomaltase synthesis as previouslydescribed (4). Between 100 and 200 recombinants wereanalyzed for each cross. Only 20 to 90o of them wereconstitutive when the AmaL45IJ deletion was used(crossover of type 2), whereas all of them were consti-tutive when the AmaLA510 deletion was used. Thisresult indicated that the malTp mutations are locatedbetween the endpoints of the two deletions (Fig. 2).

RESULTSCAP sfimulates malT expression. Strains have

been constructed in which the beginning ofmalTwas fused to an almost intact lacZ gene (2). Inthese strains the appearance of 0-galactosidaseactivity reflects the activity of the malT promot-er. That this activity was stimulated by cAMPwhen the cells were grown in glucose-minimalmedium implied that malT expression requiredthe action of CAP (2). This has now beenconfirmed by demonstrating that the introduc-tion of crp deletions in two malT-lacZ fusionstrains led to a significant reduction in theamount of 3-galactosidasis activity (Table 2).Mutants which express mafT-lacZ in the ab-

sence ofCAP. The Acrp derivatives of malT-lacZfusion strains expressed the hybrid gene (andlacY) at such a low level that they failed to growon lactose synthetic agar. Starting from fourstrains which carried either oftwo fusions (542-1or 544-5) and either of two crp deletions (39 or%), we selected mutants able to grow on thismedium. All of the mutants tested had 10 to 50times more 3-galactosidase activity than theirparental strains (not shown). As will be demon-strated (Table 3), these mutants could be dividedinto at least two classes. Mutants in the firstclass expressed malT-lacZ at the same high level

whether or not a crp+ allele was present in thestrain. Mutants in the second class synthesizedmore hybrid protein in the presence of the crp+allele. Mutants in the latter class could be identi-cal to the "up promoter" mutants selected byDebarbouilid and Schwartz in a crp+ back-ground (3).

Genetic mapping of the mutations. By analogywith results obtained with other systems (3), itseemed likely that the mutations present in theabove-described mutants were located in, orclose to, the promoter of the hybrid gene. The

TABLE 2. 1B-Galactosidase activity in malT-lacZfusion strains

a-Galactosidase activi-

maidagene crp allele0 ty (U/mg of protein)bhybrid geneGlu Glu + cAMP

542-1 Acrp-39 1.4 1.4Acrp-39/crp+ 5.0 15.0

542-1 Acrp-96 2.1 2.0Acrp-96/crp+ 8.0 20.0

544-5 Acrp-39 0.6 0.5Acrp-391crp+ 2.5 5.0

544-5 Acrp-96 1.4 1.4Acrp-96/crp+ 2.0 7.0

a Strains carrying a maIT-lacZ fusion and a Acrpallele correspond to pop3697, pop3698, pop3699, andpop3700 (Table 1). The merodiploids containing acrp+ allele were obtained by lysogenizing the abovestrains by A dcrp+.

b The specific activity of -galactosidase was deter-mined after growing the strains in minimal glucosemedium (Glu) or in the same medium with 5 mMcAMP (Glu + cAMP).

a)

b)

+

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ROLE OF CAP IN E. COLI MALTOSE REGULON 725

mapping data presented below strongly support-ed this hypothesis.

In a first set of experiments, stocks of phagePI were grown on the mutants and used totransduce to Mal' a strain carrying a mutationlocated very early in malT (Fig. 3a). If themutations were closely linked to the malT pro-moter, they had to be present in most of theMal' transductants. They would then be locatedin the cis position to a wild-type malT gene. Wetested this by reintroducing a malT-lacZ hybridgene in the cis position to the malT promoterpresent in the transductants (legend, Fig. 3b).The results shown in Table 3 (column 1) demon-strated that the Mal' transductants did contain amutation which allowed an increased expressionof malT in a Acrp as well as in a crp+ back-

a)

ground. In addition, the results showed that, asindicated earlier, the expression of the hybridgene was still controlled by CAP in some mu-tants and not in others.Once located cis to a malT+ gene, the muta-

tions often led to a partially constitutive amylo-maltase synthesis (see below). This phenotypecould be detected on individual colonies and wasused for mapping the mutations more precisely(see Materials and Methods). The result was thatall of the mutations tested recombined withdeletion 511, which ends 146 base pairs beforethe initiation codon in malT, but not with dele-tion 510, which ends early in the coding region(Fig. 2). Therefore, the mutations tested wereclosely linked to the promoter of malT. Theywere designated malTp.

roB MOM ma/P §imW cz

aroB malO malP malT

~~malT 'lacZ lbcY )

250

aroB malO ma/P malT

PI donor

recipient

I 4 1a 'a'aSAa a

aroB malQ malP l malT

12/acZ

_ ~ ~~~~~~. . .a

lacY A a 250 malTV.

FIG. 3. Transfer of the mutations in the cis position to a wild-type malT gene. The technique used wasessentially that described by Debarbouille and Schwartz (3). (a) P1 phage grown on each mutant was used totransduce an aroB malT131 recipient strain (malT131 is a 1,200-base pair insertion located earlier in the firstinterval in malT [11, 19]; malT131 is indicated by an open square, and aroB is shown by a closed square). If themutation (0) responsible for the Lac' phenotype of the mutant was closely linked to the malT promoter, thenmost of the Mal- transductants would have to carry this mutation. (b) Quasi-isogenic aroB+ crp+ and aroB+Acrp-39 derivatives of the above transductant were constructed by using P1 phage stock grown on pop 3661 andpop 3650, respectively (not shown). Verification of the presence of the mutations in these derivatives requiredthe reintroduction of the malT-lacZ hybrid gene cis to the malT promoter. This was done by lysogenizing with alambda transducing phage (first line) carrying a (malT-lacZ)542-1(Hyb) hybrid gene which contained the ambermutation malT250 (indicated by x). Phenotypic mixing with 4)80 vir was used to allow the phage to infect theAcrp-39 strains.

,m=

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TABLE 3. Effect of malTp mutations on the maltose regulonU/mg of protein

allelep crp allele p_GaJactosidaseb AmylomgltasC (malQ) Lambda receptor" (lamB)[(malT-lacZ)542-1J ML ML + MAL ML ML + MAL

Wild type Acrp-39 1 4 6 <0.01 <0.01crp+ 5 12 125 6.00 40.00

malTpl Acrp-39 56 60 142 0.10 2.60crp+ 62 37 215 28.00 27.00

malTp8 Acrp-39 67 85 327 0.70 3.00crp+ 36 41 206 39.00 54.00

malTp9 Acrp-39 70 86 125 0.10 1.80crp+ 86 47 209 40.00 69.00

malTp2 Acrp-39 16 12 82 Not done 0.90crp+ 41 3 95 22.00 50.00

malTp4 Acrp-39 Not done Not done Not done Not done Not donecrp+ 183 39 227 38.00 49.00

malTp6 Acrp-39 45 24 48 0.03 0.15crp+ 98 32 186 20.00 44.00

malTp7 Acrp-39 35 12 41 0.01 0.06crp + 215 41 174 26.00 60.00

a All of the mutations were mapped as described in the legend to Fig. 3 with the exception of malTp2.b ,-Galactosidase activity was determined in strains in which the (ma1T-1acZ)542-1(Hyb) hybrid gene has been

introduced in a cis position to the malTp mutation (Fig. 3). The strains were grown in complete medium (ML).C Amylomaltase activity was determined in strains in which the malTp mutation was located cis to a wild-type

malT gene (Fig. 3). The strains were grown in complete medium with (ML) or complete medium 0.4% maltose(ML + MAL).d Lambda receptor activity was determined in the same strains and same medium as amylomaltase. It is

expressed in thousands of molecules per bacterium.

Expression of the maltose operons In maflpstrains. In the preceding section we describedthe construction of strains bearing the malTpmutations cis to a wild-type malT gene (see Fig.3). Assuming that the malTp mutations had thesame effect on malT expression as they had onthat of malT-lacZ, these strains were expectedto synthesize high levels of MalT protein in theabsence as well as in the presence of CAP. If theonly effect of CAP in the maltose regulon hadbeen to control the level of malT expression,then the Acrp malTp strains should have grownin maltose synthetic medium. This was not thecase, even though some of the strains (thosebearing malTpl, malTp6, malTp8, or malTp9)appeared Mal' on maltose-MacConkey indica-tor medium. Thus, CAP was still required for theexpression of at least one of the mal operons,even when the MalT protein was produced inexcess. We then proceeded to investigate theeffect of the malTp mutations on the expressionof each of the operons. The assays were per-formed after growing the cells in complete medi-um (ML) because no satisfactory synthetic me-dium could be found to grow the Acrp strains.(These strains failed to use any of the commoncarbon sources except glucose, and glucose hadto be discarded because it inhibited maltosetransport and therefore prevented induction [5;A. Ullmann and A. Danchin, Adv. Cyclic Nucle-otide Res., in press].)

The expression of malK-lamB was followedby assaying the amount of LamB protein (phagelambda receptor). Three main conclusions couldbe drawn from the results (Table 3). First, thesynthesis ofLamB protein was almost complete-ly constitutive in crp+ malTp strains. A similarobservation had already been made in otherinstances in which the MalT protein was over-produced (3, 19). This constitutivity probablyresulted from the overproduction of the lowfraction of active MalT protein (P2 conforma-tion; see Fig. 1), which must be present inuninduced cells.

Second, the malTp mutations suppressed onlypartally the effect of Acrp on lamB expression.Indeed, the Acrp malTp strains contained muchless LamB protein than the wild-type strain(crp+malT+) grown under the same conditions(< 2% in the absence of maltose, 5 to 10%6 in itspresence). In these strains, the deficit in LamBprotein could not be attributed to the presence ofan insufficient amount of MalT protein (seeTable 3, first column). Therefore, CAP mustexert a direct action on malK-lamB expression,independent of its action on malT.

Third, the low-level expression of lamB ob-served in the Acrp malTp strains was maltoseinducible, whereas it was essentially constitu-tive in the corresponding crp+ strains. Thisresult was observed even when malT expressionwas the same in the crp+ and Acrp strains. It

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ROLE OF CAP IN E. COLI MALTOSE REGULON 727

suggested that higher concentrations of activeMalT product (P2 form; see Fig. 1) were re-quired to induce malK-lamB when CAP was notpresent.The expression of the malEFG operon was

followed by evaluating the amount of MalEproduct (maltose binding protein) in whole-cellextracts. This evaluation was performed afterelectrophoresis on polyacrylamide gels (Fig. 4).The synthesis of MalE protein was fully consti-tutive in the crp+ malTp strains, but very low intheir Acrp counterparts. Therefore, as was thecase with malK-lamB, the expression ofmalEFG still required CAP even when malT wasexpressed at a high level.

Ma/Ti maITfTP8 naITp7

*_.. .. .. ..~~~~~~~~~~~~~~~~~~~~~~E

... .. @~~~~~~~~~~~~~

FIG. 4. Expression of the malE gene in malTpstrains. The genotypes of the strains with respect tomalT and crp are indicated on the figure; (+) and (-)indicate that the strains carried a crp+ or a Acrp-39allele, respectively. The bacteria were grown in com-plete medium (ML) except the malT+ strains, whichwere also grown in the presence of0.4% maltose in themedium (lanes 4 and 6, from the left). The cells wereharvested at an absorbance at 600 nm of 1, centri-fuged, concentrated 40-fold in sample buffer, boiledfor 5 min, and subjected to electrophoresis on poly-acrylamide gel (9%o acrylamide) in the presence ofsodium dodecyl sulfate, as previously described (2).The maltose binding protein (MBP), a product of thegene malE, can be recognized on the gel from itsmolecular weight and the fact that it is maltose induc-ible.

The results obtained with malPQ differedmarkedly from those obtained with the twoother operons (Table 3). First, the synthesis ofamylomaltase (MalQ product) was only slightlyconstitutive in the crp+ malTp strains. This wasnot surprising since several lines of evidence hadpreviously suggested that higher concentrationsof activated MalT protein were required to stim-ulate the expression of malPQ than that of thetwo other operons (3, 25). Second, the amountof amylomaltase was not very different in thecrp+ and Acrp backgrounds. For some of themutations, the introduction of a crp+ allelecaused a slight increase in the induced level ofamylomaltase. This could merely be a conse-quence of a defect in inducer uptake, itselfresulting from the low-level expression of themalK-lamB and malEFG operons. Additionaldata in favor of the latter explanation wereobtained by using a mutation, malTIc, whichrenders the MalT protein able to activate thethree mal operons, in the absence of maltose (4).Taken by itself, this mutation did not allow theexpression of any of the mal operons in theabsence of CAP. When combined with themalTp mutation, it allowed a fully constitutiveexpression of malQ (data not shown). In conclu-sion, unlike that of the malK-lamB and malEFGoperons, the expression of malPQ is indepen-dent of CAP when a malTp mutation is present.

In the above discussion, it has been assumedthat the effect of the malTp mutation on malPQexpression was exerted in trans, through anoverproduction of MalT product. However, itmight be argued that mutations have a cis effecton malPQ expression. This was shown not to bethe case by isolating a malQ mutant from amalTp strain, lysogenizing it with a malQ+transducing phage (480 dmalA2), and demon-strating that the resulting lysogen synthesizedamylomaltase at a low constitutive level (datanot shown).

DISCUSSIONResults presented herein and previously (2, 3)

demonstrated that malT expression is stimulatedby CAP and cAMP. Knowing that the MalTprotein is present in limiting amounts in wild-type cells (2, 9), it seemed possible that CAPcontrolled the expression of the mal operonssimply by modulating the level of malT expres-sion. In this work we described mutations(malTp) most probably located in the promoterregion of the gene malT and which allowed thisgene to be expressed at a very high level even inthe absence of CAP. In the presence of thesemutations the malK-lamB and malEFG operonsstill required CAP to be fully expressed. There-fore, the action of CAP on these operons cannotbe simply explained by a modulation of malT

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728 CHAPON

expression. On the other hand, the expression ofthe malPQ operon became independent of CAPwhen a malTp mutation was present. This couldbe taken as evidence that the effect of CAP onthis operon is only a consequence of its effect onmalT expression. However, it should be recalledthat the malTp cells probably contained signifi-cantly more MalT protein than did the wild-typecells. Conceivably, malPQ expression may re-quire the action of CAP under normal condi-tions, but this action may become unecessarywhen the MalT protein is present in excess.

Guidi-Rontani and Gicquel-Sanzey recentlydescribed mutations, probably located in themalT region, that bear some similarities withthose described herein (8). However, these mu-tations allowed Acrp strains to grow on syntheticmaltose medium and, more important, most didnot lead to a constitutive expression of themaltose operon in a crp+ background. They arethen clearly different from the malTp mutations.They could affect the structure of the MalTprotein in such a way that it stimulates theexpression of the mal operons even in the ab-sence of CAP.

All known facts concerning the interplay ofCAP and the MalT protein in the maltose regu-lon could be assembled in the following model.

(i) CAP and cAMP would enhance the affinityof the MalT protein for the promoter region ofthe malEFG and malK-lamB and possibly of themalPQ operons. A similar hypothesis based onextensive in vitro studies was proposed to ac-count for the action of CAP on the araBADoperon (12, 15).

(ii) In the absence of CAP, the affinity of theMalT protein for the malPQ promoter would behigher than that for the two other promoters.This would account for the fact that the malPQoperon, unlike the two others, is fully expressedin Acrp malTp strains.

(iii) In the presence of the CAP-cAMP com-plex, the affinity of the MalT protein for themalK-lamB and malEFG promoters would behigher than that for the malPQ promoter. Thiswould account for the observation that, in crp+strains, the expression of malK-lamB andmalEFG requires less MalT protein than doesthat of malPQ (3, 25; this work).

This model should be amenable to testing invitro since most of the elements required toperform the experiments are now available. Po-tential binding sites for CAP and the MalTprotein have already been recognized in thepromoter regions of the three operons (M. De-barbouilld, P. Cossard, and 0. Raibaud, Mol.Gen. Genet., in press; H. Bddouelle and M.Hofnung, Mol. Gen. Genet., in press). The mainlimitation is obtaining the MalT protein in anative form (19; Ddbarbouilld et al., in press).

The controls exerted by CAP and the MalTprotein have different functions at the physiolog-ical level. There is evidence that the intracellularconcentration of cAMP is controlled by theextracellular concentration of glucose or of oth-ers sugars (PTS sugars) taken up by the phospho-transferase system (5, 6, 17). The transport ofthese sugars would result in an inhibition ofadenylate cyclase and therefore in a reduction inthe internal concentration of cAMP (5; Ullmannand Danchin, in press). Under such conditionsthe malK-lamB and malEFG operons would beexpressed at a very low level and the cells wouldbe unable to transport maltose and maltodex-trins. When glucose and the others PTS sugarswould become exhausted, the intracellular levelofcAMP would rise (6) and the expression of thetwo operons involved in maltose transportwould be stimulated, whereas that of the malPQoperon would remain very low. However, ifmaltose were present in the medium, it wouldthen be transported and induce the expression ofthe malPQ operon. The primary function ofCAP in the maltose regulon would thus be tocontrol the level of maltose transport in re-sponse to the presence of other metabolizablesubstrates, whereas the role of the MalT proteinwould be mainly to induce the synthesis of thenecessary catabolic enzymes when maltose ormaltodextrins are present.

ACKNOWLEDGMENTSI thank M. Debarbouill6, who constructed the maiT-lacZ

fusion strains. I am especially indebted to Olivier Raibaud andMaxime Schwartz for their constant interest in this work andfor their help in preparing the manuscript.

This work was supported by grants from the Centre Nation-al de la Recherche Scientifique (LA 270) and the DGRST (80 701 82).

ADDENDUM IN PROOFThe activities of ,B-galactosidase reported in Tables

2 and 3 should be multiplied by 4 to obtain the correctunits per miligram of protein.

LITERATURE CITED1. Braun, V., and H. J. Krieger-Brauer. 1977. Interrelation-

ship of the phage A receptor protein and maltose transportin mutants of Escherichia coli K12. Biochim. Biophys.Acta 469:89-98.

2. Dfbarboufle, M., and M. Schwartz. 1979. The use of genefusions to study the expression of malT, the positiveregulator gene of the maltose regulon. J. Mol. Biol.132:521-534.

3. DEbarbouilIW, M., and M. Schwartz. 1980. Mutants whichmake more malT product, the activator of the maltoseregulon in Escherichia coli. Mol. Gen. Genet. 811:1-7.

4. Ddbarbouilld, M., H. A. Shuman, T. J. Silhavy, and M.Schwartz. 1978. Dominant constitutive mutations in malT,the positive regulator gene of the maltose regulon inEscherichia coli. J. Mol. Biol. 124:359-371.

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