Vol. 141, No. 2JouRNAL OF BACTERIOLOGY, Feb. 1980, p. 751-7570021-9193/80/02-0751/07$02.00/0

Mutation in the crp Gene of Salmonella typhimurium WhichInterferes with Inducer Exclusion

B. J. SCHOLTE AND P. W. POSTMA*Laboratory ofBiochemistry, B. C. P. Jansen Institute, University ofAmsterdam, 1018 TVAmsterdam, The

Netherlands

A mutation in the crp gene of Salmonella typhimurium is described whichovercame the defects ofptsHI deletion mutants for growth on a number of non-phosphotransferase system compounds. This mutation abolished inducer exclu-sion in a leaky ptsI mutant. The possible implications for the mechanism ofinducer exclusion are discussed.

The gram-negative bacteria Escherichia coliand Sabnonella typhimurium contain a phos-phoenolpyruvate-dependent sugar phospho-transferase system (PTS) which is active in thetranslocation and concomitant phosphorylationof a number of sugars (15). The PTS consists ofa number of protein components which catalyzethe transfer ofphosphoryl groups from phospho-enolpyruvate to the sugar (Fig. 1). In addition totheir role in transport, some of these proteinshave also been implicated in the regulation ofcell metabolism. In particular, glucose-specificfactor m (I1G1) or a closely related regulatoryprotein is thought to play a central role (15, 17,20). The regulatory role of the PTS is illustratedby the pleiotropic nature ofmutants defective inone or more components of the PTS. E. coli orS. typhimurium mutant strains which lack en-zyme I or HPr or both do not grow on a numberofnon-PTS sugars, including glycerol, melibiose,maltose, and lactose (see Table 2; for a review,see reference 15). In E. coli this phenomenonhas been attributed mainly to a low rate of cyclicAMP (cAMP) synthesis. There is evidence thatthe PTS is involved in regulation of adenylatecyclase activity, as suggested by the inhibitionof adenylate cyclase activity by PTS sugars (7).Phosphorylated enzyme I and a phosphorylatedregulatory protein have been proposed as acti-vators of adenylate cyclase (13, 17).For S. typhimurium, on the other hand, the

inability of pts mutants to grow on many non-PTS carbon sources has been explained mainlyon the basis of inducer exclusion (15, 18-20).The non-phosphorylated form of a protein com-ponent of the PTS is thought to inhibit thetransport systems for glycerol, melibiose, andmaltose, preventing the entry ofthese substrates(inducers). It has been proposed that the pri-mary effector of this inhibition is mGlc, a proteincomponent of the PTS involved in transport ofglucose via enzyme Hl-BGc (Fig. 1). Support for

this proposal has been provided by the isolationof crr mutants. The crr mutation lowers theactivity of IIIGiC in the cell and at the same timerestores growth of pts mutants on several non-PTS carbon sources (19; see also Table 2). Theconcept of this type ofinducer exclusion predictsthat inhibition of the entry of inducer preventsinduction and subsequent growth, irrespectiveof the presence of cAMP. In other words, ifinducer exclusion is involved in the growth in-hibition ofpts mutants on melibiose, glycerol, ormaltose, one would not expect that cAMP wouldrestore growth of these pts mutants.

In this paper we report that either cAMP ora mutation in the cAMP binding protein (crp*)is able to overcome the effect of pts mutationsin S. typhimurium with respect to growth onsome non-PTS compounds. In particular, ourresults show that inducer exclusion in ptsI mu-tants is abolished either by the addition ofcAMP or by the introduction of a crp* mutation.The consequences for the current concept ofinducer exclusion are discussed.

MATERIAlJS AND METHODSBacterial strains. The strains of S. tyhunurium

used in this study are listed in Table 1. The phenotypiccharacteristics of representative strains are describedin Table 2.Media and growth conditions. Cells were grown

at 370C on a rotary shaker in liquid medium A [con-taining, per liter of distilled water: (NH4)2SO4, 1 g;K2HPO4, 10.5 g, KH2PO4, 4.5 g, MgSO4, 0.1 g] supple-mented with 20 pg of tryptophan per ml and a carbonsource (0.2%). Media were solidified with agar (1.5%;Difco Laboratories, Detroit, Mich.). The effect ofcAMP was tested in two ways: (i) by adding 5 mMcAMP to the liquid growth medium and followinggrowth by measuring the increase in optical density at600 nm; (ii) by placing a sterile filter with 5 pmol ofcAMP in the middle of an agar plate and recordingthe diameter of the growth zone after 24 to 48 h.Transductions involving TnlO were performed on nu-trient agar plates (0.8% nutrient broth, 0.5% NaCl, and

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752 SCHOLTE AND POSTMA

Sugar

PEP EnzYM I4 P-HiPr

A n~~~~~AP

Ad"nyate

Sugar-P

Sugar

FIG. 1. The phosphoenolpyruvate-dependent PTSconsists of two general protein, enzyme I and HPr

and a nwnber of sugar-specific proteins. Here are

shown msconsisting of Il-A /IIB and I exclusion is indicated

by. the inhibition (-) of non-phosphotransfera-se

transport systems (S represents melibiose, maltose-or

glycerol) by non-phosphorylated IIIG'. Activation

of adenylate cyclase by phosphorylated HIIGl is

also shown.

1.5% agar) containing 25jpg of tetracycline per ml.

.Chemicals. [U-11CJglycerol. (46 mCi/mmol), [U-14C]methyl a-glucoside (184 mCi/mmol), (2,8-3H]-cAMP (52 Ci/MMOl), and [U_,4C]glUCOSe(284 MCi!

mmol) were obtained f3rom The Radiochemical Centre,

Amersham, England. cAMP was purchased from

Sigma Chemical Co., St. Louis, Mo.

Preparation of cell extracts. Cell were grown

overnight nutrient broth. After being washed with

0.9% NaCl, the cells were suspended in a buffer con-

ta.ining 25 mM potassium phosphate, 10 mM MgC12,0.1 mM EDTA, and 0.5mM dithioerythritol (final pH,

7.5) and were broken by passage through an Aminco

French pressure cell at 1,200 kg/cm2. The homogenate

was centrifuged for 20 mmn at 12,000 x g and 49C to

remove intact cells and debris. The resultming cell ex-

tract was centrifged for 120 mmn at 100,000 X g and

yielded a clear supernatant which was dialyzed over-

night against three changes ofbuffer.t Protein was

determiined by, the method of Lowry et al. (9).

Determinati'on of bning activity frcAMP.

indig oH]cAMP to the cAMP binding proteinwas determined in the dialyzed supernatant essentially

as described by Pastan et al. (11), with the following

modifications. The incubation mixture contained 10

mM AMP, 10 mM potassium phosphate buffer

7.5), various amounts of [3H]cAMP (specific-activity,2,300 cpm/pmol), and 10 tof20 mg of protein per ml.

[4C]glucose (10 mM) was added to correct for non-

boundwH included in the pellet.

Tranport and oxidation studies. Growth of cells

and tranport of labeled compounds were as desfribedpreviously (14). Trasmport rate is expred as nano-

moles of substrate taken up per minute per miir

J. BACTERIOL.

(dry weight) at 200C. Oxygen consumption was meas-ured with a Clark-type electrode in medium A (finalvolume, 1.6 ml-). Substrates were added at the concen-trations indicated below. The oxidation velocity isexpressed as nanoatoms of oxygen consumed per min-ute per milligram (dry weight) at 25°C.

Isolation of mutants and genetic methods.Strains containing only a crr mutation were con-structed as follows. A AptsHI41 crr double mutant wastransduced with phage P22 grown on strain SB3687[A(pt8I-crr)166, see reference 4], with selection forpt8+ recombinants (growth on mannitol). SinceApt8HI41 ends in the middle of the pt8l gene (5) andA(ptsI-crr)166 starts at the end of the pt8I gene (4),pts8 recombinants can be obtained which still containthe crr mutation. The properties of these crr strainswill be reported elsewhere. Briefly, they do not growon a number ofnon-PTS compounds, including xylose,citrate, succinate, and malate (Table 2). Revertants ofthe crr mutants which regained the Crr+ phenotypecould be obtained on minimal plates containing xyloseor citrate as the carbon source, either spontaneouslyor upon addition of diethyl sulfate. One class of re-vertants contained reversions that mapped near cysAand represented true crr+ reversions. A second classof revertants contained a mutation cotransduciblewith cysG which was subsequently named crp*. Astrain containing only this crp* mutation was con-structed by replacing the crr-306 mutation in a crpcrr strain by the ptsHI-crr-49 deletion. This deletionextends beyond the crr-306 mutation. PP869 wastransduced with phage P22 grown on PP866, withselection for both tetracycline resistance and inabilityto grow on mannitol. Transduction of the resultingstrain with wild-type phage to growth on mannitol inthe absence of cysteine yielded the crp strain PP914.In an analogous way, crp ptsIl 7 mutants were iso-lated. Preparation of P22 transducing lysates andtransduction with phage P22 were performed as de-scribed previo"isly (2).

RESULTS

Effect ofcAMP onpts mutants. The modelof inducer exclusion as described above predictsthat strains carrying a ptsHI deletion mutationcannot grow on non-PTS carbon sources such asglycerol, melibiose, or maltose as long as 1G1c ipresent in the non-phosphorylated form. Underthese conditions, the transport of these carbonsources is inhibited. We have found, however,that cAMP, added externally, stimulated thegrowth of ptsHI deletion mutants of S. typhi-murium on glycerol and melibiose (but not onmaltose) (Table 2). For instance, PP642(AptsHI41) doubled on glycerol every 75 minwhen 5 mM cAMP was added to the medium.In the absence of cAMP, no growth was ob-served. Since in these strains GIIPc is supposedto be completely in the non-phosphorylatedform due to the absence of enzyme I and HPr,one might argue that an alternative route forphosphorylation of IlIGIC, independent ofenzyme

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crp MUTATION IN S. TYPHIMURIUM 753

TABLE 1. Origin and genotype of Salmonella strainsa

Strain Relevant genotype Isolation procedure, parental SourceStram Relevantgenotype ~~~~~ ~ ~~~~strainSucSB3507 trpB223 E. BalbinderSB2309 trpB223 A (cysK-ptsHI)41 J. C. CordaroPP386 crr-306 A(cysK-ptsHI)41 trpB223 Glp+ SB2309, spontaneous This studyPP776 crr-306 trpB223 PP386 x P22(SB3687) This studyPP782 crr-306 cysA20 x P22(PP776) This studyPP825 crp*-771 crr-306 Xyl+ PP782, DES This studyPP838 cysG1510:.:Tn10 crr-306 PP782 x P22(TT172) This studyPP869 crp*-771 crr-306 PP838 x P22(PP825) This studyPP914 crp*-771 This studyPP642 A(cysK-ptsHI)41 cysA20 x P22(SB2309) This studyPP866 A(cysK-ptsHI-crr)49 cysA1539.:Tn1O trpB223 This studyPP712 cysG1510.:Tn10 A(cysK-ptsHI)41 PP642 x P22(TT172) This studyPP886 cysG1510 :TnlO A(cysK-ptsHl)41 trpB223 This studyPP889 crp*-771 A(cysK-ptsHI)41 trpB223 PP886 x P22(PP825) This studySB1476 ptsIl7 P. E. Hartman

crp*-771 ptsIl 7 This studySB1786 cya-502 P. E. HartmanPP958 cya-502 crp*-771 This study

cysA20 P. E. HartmanSB3687 A(ptsI-crr)167tpB223 P. E. Hartman,

ref 4TT172 cysG1510:.:TnlO J. RothNK186 cysA1539-.:TnlO J. RothTA3335 crp B. N. Ames, ref.

1a Genetic nomenclature according to Sanderson and Hartman (21). Glp, Glycerol; Xyl, xylose; DES, diethyl

sulfate; P22, phage P22.

TABLE 2. Phenotype of strains used in this studya

cAMP Growth'Genotype presentb Mtld Glp Mel Malt Xyl

Wild type - + + + + +AptsHI - -

AptsHI + - + + - +AptsHI crp* - - + + - +AptsHI crr - - + + + -

crr - + + + + -

crr + + + + + +crr crp * - + + + + +crp * - + + + + +

aMtl, Mannitol; Glp, glycerol; Mel, melibiose; Malt,maltose; Xyl, xylose.

b If cAMP was present, 5 ,umol was added.'Growth was monitored on chemically defined me-

dia containing 0.2% (wt/vol) of the carbon source, andfermentation was tested on eosin methylene blueplates containing 1% sugar. +, Growth and fermenta-tion after 48 h of incubation at 37°C; -, no growth andno fermentation under these conditions.

d Phenotype.

I and HPr, is created when these mutants aregrown in the presence of cAMP. This wouldallow transport of glycerol and melibiose andsubsequent growth on these carbon sources. Theexperiments shown in Fig. 2 eliminated this pos-sibility. Figure 2A shows an example of inducer

exclusion as described earlier by Saier and Rose-man (20). The PTS sugar methyl a-glucosideinhibits glycerol oxidation in a leaky ptsI mu-tant. The rapid inhibition is explained by themechanism of inducer exclusion: all of the phos-pho-LIIGlc present in the cell is dephosphorylatedrapidly by methyl a-glucoside via its interactionwith sugar-specific enzyme II-BGlc (see also Fig.1). Figure 2B shows that glycerol oxidiation bya ptsHI deletion mutant, grown on glycerol inthe presence of cAMP, is not sensitive to inhi-bition by methyl a-glucoside. This indicates thatin ptsHI deletion strains, grown under theseconditions, inducer exclusion was not effective,since any phospho-IIIGlc formed by the alterna-tive pathway would be dephosphorylated bymethyl a-glucoside via enzyme II-BGlc.Mutations which modify the cAMP bind-

ing protein. We found other evidence indicat-ing that cAMP is in some way involved in in-ducer exclusion. As described by Saier and Rose-man (19), growth defects ofpts mutants on var-ious non-PTS compounds can be suppressed bya crr mutation. In these pts crr double mutants,the crr mutation results in a lowered IIIG1c level.Mutants containing only a crr mutation wereconstructed as described in Materials and Meth-ods. These crr mutants did not grow on a num-ber of non-PTS compounds, including xylose,

VOL. 141, 1980

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754 SCHOLTE AND POSTMA

© ©

\\\\\\M\\\ \o\M MG

FIG. 2. Influence of methyl a-gluco8ide (aMG) onglycerol oxidation. Cells weregrown on minimal saltsmedium and the carbon source indicated below. 02consumption was measured as described in the text.The reaction wa8started by the addition of 6 mMglycerol (at the first arrow). At the second arrow, 1mM methyl a-glucoside was added. The dotted linein A represents oxidation in the absence of methyla-glucoside. (A) SB1476ptsIl7grown on 0.2%glycerol(1.8mg dry weightper ml). (B) PP642 A(cysK-ptsHI)41grown on 0.2% glycerol plus 5mM cAMP (0.5 mg dryweight per ml). (C) ptsIl7 crp*-771 grown on 0.2%glycerol (0.6 mg dry weightper ml).

citrate, succinate, and malate, but grew nornallyon all PTS sugars or on the non-PTS sugarsglycerol, maltose, and melibiose (Table 2). Lackof growth on xylose, citrate, or succinate couldbe overcome by the addition of external cAMP(Table 2). For instance, PP782 (crr-306) doubledevery 60 min on xylose in the presence of 5 mMcAMP, whereas no growth occurred in the ab-sence of cAMP. Starting with these crr strains,we isolated suppressors of the crr mutationwhich allowed, in addition, pts mutants to growon a number of non-NTS carbon sources. Re-vertants of crr strains were sought which re-gained the ability to grow on xylose, citrate, andsuccinate at the same time. Some of these re-vertants carried a mutation which mapped closeto cysA and were shown to have regained iGicactivity. These stains presumably acquired re-versions in the crr gene, correcting the originalcrr mutation. A second class of revertants alsoexhibited the Crr+ phenotype, but the mutationdid not map near cysA. Subsequent mappingshowed that these mutations were localized closeto cysG, being 15 to 30% cotransducible withcysGl5l1Y.:TnlO (a strain containing a TnlOtransposon inserted in cysG). For instance, whenPP838 (crr-306 cysG1510:.:Tn1O) was transducedwith phage P22 grown on PP825 (crp*-771) onminimal plates lacking cysteine, the resultingcysteine+ transductants (43 colonies) fell intotwo classes: 14% of the transductants grew oncitrate, succinate, and xylose, whereas 86% wereunable to do so. Both classes tained loweredIfflGc activity, indicating that the original crrmutation was still present. Several lines of evi-dence suggest that this suppressor mutation was

probably in the crp gene, which codes for thecAMP binding protein. (i) Hong et aL (8) haveshown that crp is 16% cotransducible withcysG439. (ii) The suppressor mutation can par-tially restore growth defects of cya mutants, i.e.,mutants that lack adenylate cyclase and conse-quently are unable to grow on many carbonsources (10, 12). Strains containing both cya andthe suppressor mutation (for instance, PP958cya-502 crp*-771) are able to grow again on, forinstance, mannitol or melibiose, carbon sourceson which the cya strain does not grow (see alsoreference 22). (iii) Figure 3 shows that this sup-pressor mutation caused a decrease in the bind-ing affinity for cAMP as measured in a cellextract. The strain used was constructed bytransducing the suppressor mutation into a cysGstrain, eliminating the possibility that more thanone mutation was involved. From these data weconclude that the suppressor mutation was in

c-AMP concentration ( jM)

E

E0.

10-~~~

.8A

u

0 1 2 3

B 0.4-E00..E~ 03-3

0.2.8

01.

0 21/c-AMP concentration (jM)

FIG. 3. Binding affinity for cAMP in crude ex-tracts. The points in the figures represent the meanof three determinations with the same preparation.(A) Binding of cAMP at different concentrations ofcAMP; values are calculated as picomoles per milli-gram of protein. -(B) Double-reciprocal plot of thedata of (A). The values for crp * and crp are cor-rected for aspecific binding by subtracting the valuesfor the crp strain. Symbols: 0, crp' (SB3507); *, crp(PP914); A, crp (TA3335).

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VOL. 141, 1980

the crp gene; it will be designated crp* hereafter.Strains carrying only the crp* mutation, for

instance PP914, showed nonnal growth on allcarbon sources tested (Table 2), indicating thatthe cAMP binding protein was still functionalalthough it had altered binding properties.Strains with a defective cAMP binding proteininstead do not grow on any of these carbonsources (6, 10).

Revertants have been isolated from severalcrr strains. Until now, in all such cases, thesuppressor mutation was cotransducible witheither cysG or cysA.Effect of crp* on pts mutants. In view of

the observed effects of cAMP on pts mutants,we decided to test whether the crp* mutationalso affected the growth of pts mutants. Thefollowing results were found. (i) A crp* AptsHIdouble mutant was able to grow on glycerol ormelibiose but not on maltose (Table 2). Theseresults resemble those from experiments withexternally added cAMP. Oxidation of glycerol ormelibiose was not inhibited by methyl a-gluco-side in these strains, results similar to thoseobtained with these strains grown in the pres-ence of cAMP. (ii) A strain carrying both theleaky ptsI17 mutation and a crp* mutationshowed no inducer exclusion as measured by theeffect of methyl a-glucoside on both glyceroloxidation (Fig. 2C) and glycerol transport (Fig.4). Similar results were obtained when thio-methyl galactoside transport via the melibiosetransport system was measured (data notshown).

0 1 2 0 1 2Time (min)

FIG. 4. Inducer exclusion in a crp strain. Cellswere grown on a minimal salts medium containing0.2% glycerol. Transport of 1 mM [U- 14CJglycerol(specific activity, 110 cpm/nmol) was measured asdescribed in the text. (A) SB1476: *, no methyl a-glucoside; 0, 1 mM methyl a-glucoside. (B) crp *771ptsII7: *, no methyl a-glucoside; 0, 1 mM methyl a-glucoside.

crp MUTATION IN S. TYPHIMURIUM 755

The crp* mutation did not abolish inducerexclusion by interfering with the synthesis ofIIIGI,. Transport ofmethyl a-glucoside, a specificsubstrate of the IIlGc/II-BGlc system, was normalor even elevated in crp* strains compared withthat in the crp+ parent (Fig. 5). Finally, the crp*mutation did not result in constitutive synthesisof the transport system for glycerol or melibiose,as illustrated for glycerol in Fig. 6. Cells grownin minimal medium with galactose as a carbonsource did not take up glycerol, whereas growthon glycerol induced normal uptake. This meansthat there was a normal requirement for theinducer in the crp* strain.

DISCUSSIONRegulation of bacterial metabolism by the

phosphoenolpyruvate-dependent PTS is a com-plex phenomenon. It has been proposed thatIIIGkc or a closely connected regulatory proteininteracts with a number of non-sugar phospho-transferase transport systems and adenylate cy-clase (15,17). Non-phosphorylated 111Gkc inhibitsthese transport systems (inducer exclusion),whereas phosphorylated IIIGk activates adenyl-ate cyclase. Although this scheme can accountfor a number of observations, two difficultiesbecome clear upon closer inspection of thismodel. (i) crr mutations abolish inducer exclu-sion inpts mutants by eliminating non-phospho-rylated IIIc, and allow growth ofpts crr doublemutants on non-PTS compounds such as meli-biose, maltose, glyceroL and lactose. However,since growth on these carbon sources requires in

L-oCL$-

0n

a'a

0

I3

.d

cm

0 30Time (sec)

60

FIG. 5. Transport ofmethyl a-glucoside. Cells weregrown on minimal salts medium containing 0.2%glucose and harvested in the mid-exponential phase.Transport of 0.25 mM methyl a-[U- 14C]glucoside(specific activity, 1,400 cpm/nmol) was measured asdescribed in the text. Symbols: 0, crp+ (SB3507); ,crp* (PP914).

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,, , for activity or none at all. However, in both casesthe mechanism by which cAMP and the crp*gene product influenced inducer exclusion is asyet unclear. The prompt inhibition of glyceroloxidation or transport in ptsIl7 strains by a PTSsubstrate (Fig. 2A and 4) cannot be explained bya mechanism acting only at the level of operontranscription.These results urge us to combine inducer ex-

clusion and regulation by cAMP and crp* in onemodel. Apparently, the crpj mutation andcAMP influence one of the conditions necesaryfor the occurrence of inducer exclusion. Themost obvious explanation, namely that the crp*mutation interferes with the synthesis of thePTS proteins, in particular IlHc, can be rejectedon the basis of the evidence presented in Table

4 2 and Fig. 5. There are several other possibilities

° 30 60 90 which have to be investigated. One of these isdirect involvement of cAMP in inducer exclu-

Time (sec) sion. Another is modulation of the hypotheticalInduction of glycerol uptake. Cells were regulatory activity of IIiGi by cAMP. Possibly,d the uptake of I mM [U. "4Cjglycerol (spe. cAMP and the crp* mutation inhibit during cellity, 110 cpm/nmol) was measured as de- growth the formation of a protein other thanthe text. Symbols: 0, crp+ strain (SB3507) mGic that is essential to inducer exclusion. Fi-02% glycerol; 0, crpi strain grown on 0.2% nally, an important parameter which is possiblyA, crp* strain (PP914) grown on 0.2% influenced by the crp* mutation might be thec,crp* strain (PP914) grown on 0.2%galac- relative amounts of the various non-phospho-

transferase transport systems which are affectedcAMP (cya and crp mutants exhibit by inducer exclusion, and IVllc. This might de-f*h nn thaa %n-scarn rgRi 0 198 it. terminie the extent of the inhibition.

UXItSLlVE W6llUll t11UMzsgVzL, 1V, 1xJp, it,

is surprising that these pts crr mutants grow atall on these carbon sources since they lack phos-pho.IIlG and thus are unable to activate ade-nylate cyclase. (ii) Conversely, the earlier findingthat growth of pts mutants of E. coli on thesenon-PTS compounds can sometimes be stimu-lated by cAMP (for a review, see reference 15)is equally surprising. Although in this case thelow intracellular cAMP concentration, due tothe absence of phospho-EIGk, is corrected, in-ducer exclusion is still existent.

In this paper we present data that shed somelight on these difficulties. A mutation, crp*, isdescribed that allowed pts mutants to grow onthese non-PTS sugars. Surprisingly, the crp *mutation was isolated as a suppressor of a crrmutation which, by itself, is a suppressor ofptsmutations. The results led us to the conclusionthat the crp* mutation interfered with inducerexclusion in some way. The effect of the crp*mutation may be explained by assuming that itstimulated the adenylate cyclase activity. Theinvolvement of the crp gene in the regulation ofcAMP production and adenylate cyclase synthe-sis has been reported (3, 16). Alternatively, thecrp* mutation could alter the cAMP bindingprotein in such a way that it needed less cAMP

ACKNOWLEDGMEDGSWe thank J. Roth (University of Utah) and B. N. Ames

(University of California) for the generous gift of strains.This study was supported by a grant from The Netherlands

Organization for the Advancement of Pure Research (Z.W.O.)under the auspices of The Netherlands Foundation for Chem-ical Research (S.O.N.).

LITERATURE CITED1. Alper, M. D., and B. N. Ames. 1978. Transport of

antibiotics and metabolite analogs by systems undercyclic AMP control: positive selection of Salmonellatyphimurium cya and crp mutants. J. Bacteriol. 133:149-157.

2. Blume, A. J., and E. Balbinder. 1966. The tryptophanoperon of Salmonella typhimurium. Fine-structureanalysis by deletion mapping and abortive transduction.Genetics 53:577-592.

3. Botsford, J. L., and M. Drexler. 1978. The cyclic 3',5'-adenosine monophosphate receptor protein and regu-lation of cyclic 3',5'-adenosine monophosphate synthe-sis in Escherichia coli. Mol. Gen. Genet. 165:47456.

4. Cordaro, J. C., T. Melton, J. P. Stratic, M. Atagiin, C.Gladding, P. E. Hartman, and S. Roseman. 1976.Fosfomycin resistance: selection method for intemaland extended deletions of the phosphoenolpyruvate:sugar phosphotransferase genes of Salnonella typhi-mwium. J. Bacteriol. 128:785-793.

5. Cordaro, J. C., and S. Roseman. 1972. Deletion map-ping of the genes coding for HPr and enzyme I of thephosphoenolpyruvate:sugar phosphotransferase systemin Salmonella typhimurium. J. Bacteriol. 112:17-29.

-

a)

_x

%

3o c'i- EU 0

->%aEc

FIG. 6. .growl; azucific activiscribed ingrown on (galactose;glycerol; Atose.

all casesAmfiv-+,ua

756 SCHOLTE AND POSTMA J. BACTERIOL.

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crp MUTATION IN S. TYPHIMURIUM 757

6. Emmer, M., B. de Crombrugghe, I. Pastan, and R.Perlman. 1970. Cyclic AMP receptor protein of E. coli:its role in the synthesis of inducible enzymes. Proc.Natl. Acad. Sci. U.S.A. 66:480-487.

7. Harwood, J. P., C. Gazdar, C. Prasad, A. Peterkof-sky, S. J. Curtiss, and W. Epstein. 1976. Involvementof the glucose Enzymes II in the regulation of adenylatecyclase by glucose in Escherichia coli. J. Biol. Chem.251:2462-2468.

8. Hong, J. S., G. R. Smith, and B. N. Ames. 1971.Adenosine 3':5'-cyclic monophosphate concentration inthe bacterial host regulates the viral decision betweenlysogeny and lysis. Proc. Natl. Acad. Sci. U.S.A. 61:2258-2262.

9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein determination with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

10. Pastan, I., and S. Adhya. 1976. Cyclic adenosine 5'-monophosphate in Escherichia coli. Bacteriol. Rev. 40:527-551.

11. Pastan, I., M. Gallo, and W. B. Anderson. 1974. Thepurification and analysis of mechanism of action of acyclic AMP-acceptor protein from Escherichia coli.Methods Enzymol. 38:367-376.

12. Perlnan, R. L., and L. Pastan. 1969 Pleiotropic defi-ciency of carbohydrate utilization in an adenyl cyclasedeficient mutant of Escherichia coli. Biochem. Bio-phys. Res. Commun. 37:151-157.

13. Peterkofsky, A., and C. Gazdar. 1975. Interaction ofEnzyme I of the phosphoenolpyruvate:sugar phospho-transferase system with adenylate cyclase of Esche-richia coli. Proc. Natl. Acad. Sci. U.S.A. 72: 2920-2924.

14. Postma, P. W. 1977. Galactose transport in Salmonella

typhimurium. J. Bacteriol. 129:630-639.15. Postma, P. W., and S. Roseman 1976. The bacterial

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