Proc. Nad. Acad. Sci. USAVol. 82, pp. 4620-4624, July 1985Biochemistry

Regulatory components in Citrobacter freundii ampC(8-lactamase induction

(ampR gene/cephalosporinase/gene regulation/repressor/antibIotic resistance)

FREDERIK LINDBERG, LENNART WESTMAN, AND STAFFAN NORMARKDepartment of Microbiology, University of UmeA, S-901 87 Umei, Sweden

Communicated by Peter Reichard, March 18, 1985

ABSTRACT Citrobacter freundU encodes an induciblechromosomal P-lactamase similar to the constitutively ex-pressed ampC .8-lactamase of Escherichia coli. In the latterspecies the ampC gene is located next to the fumarate reductase(frd) operon, whereas in C. freundii the ampC gene is known tobe separated from frd by 1100 base pairs. This interveningDNA segment carries a gene, ampR, coding for a 31-kilodaltonpolypeptide. The cloned C. freundii OS60 ampC gene isinducible by j-lactam antibiotics in E. coi, but only in thepresence of an intact ampR gene. In the absence of inducer theAmpR protein represses C. freundii ampC synthesis 2.5-fold.Addition of fl-lactams induced expression from the clonedampC 13-lactamase gene 11-fold. Thus, the AnmpR protein hasa positive effect on ampC expression in the presence of inducingg3-lactams. Two spontaneous mutants of C. freundu wereisolated that constitutively overproduce the ampC g-lactamase.The mutations in both these strains occurred outside thefrd-amp region, suggesting that there is at least one additionalcomponent in the regulatory system. With the cloned C.freundiiampC gene inE. coli, mutants with the same phenotypecould be obtained. These mutations were located on the E. colichromosome. The constitutive P-lactamase overproduction inthese mutants requires the presence of an intact ampR gene.

On the basis of amino acid sequencing and enzymaticanalyses, three evolutionary classes of B-lactamases, A, B,and C, have been distinguished (1, 2). Most Gram-negativeenterobacteria express chromosomally encoded ,B-lacta-mases of class C, which act preferentially on cephalosporins(2-4). In Escherichia and Shigella this chromosomal ,B-lactamase is expressed at a low level, which is not affected bythe presence of /-lactams (5).Most species belonging to the Citrobacter, Enterobacter,

Serratia, and Pseudomonas genera, as well as indole-positiveProteus species, have a 4-lactam-inducible enzyme (5-7).The introduction of third-generation cephalosporins has ledto the unexpected increase of resistance to ,3-lactams inmembers of these genera. Resistance is due to frequentmutations leading to high constitutive production of thechromosomal ampC P3-lactamase (8, 9). This enzyme acts byeither hydrolyzing or by simply binding the ,B-lactam thatreaches the periplasmic space (10-12).The ampC gene of Citrobacter freundii has been cloned

and was shown to be closely linked to the frd operon, whichcodes for the fumarate reductase complex (4), as is the casein Escherichia coli (13). In C. freundii, however, there is an1100-base-pair (bp) DNA segment, not present in E. coli, thatseparates ampC from frd (4). We show that this segmentcodes for a trans-acting 31-kilodalton protein, AmpR, whichis responsible for the inducibility of,-lactamase synthesisfrom the C. freundii ampC gene, and that it can have both a

positive and a negative effect on ampC expression. Wefurther show that mutations leading to high constitutive levelsof ampC f3-lactamase production map outside theampR-ampC region.

MATERIALS AND METHODS

Media and Growth Conditions. L medium (14) was used forroutine purposes. M9 medium (15) containing 0.2% glucose,0.2% casamino acids, uracil at 50 ,ug/ml, and thiamin at 1,ug/ml (M9CA) was used in the determination of ampicillinresistance and .-lactamase expression. When necessary, themedia were supplemented with ampicillin (concentration asindicated), tetracycline (10 ,ug/ml), or chloramphenicol (10Ag/ml).

Determination of Ampicillin Resistance and Relative ,-Lactamase Production. Ampicillin resistance on M9CA me-dium was determined as the LD50- i.e., the concentration ofampicillin required to inhibit 50% of the single cells fromforming colonies (16). To determine the relative production of,B-lactamase, cells were washed twice (50 mM potassiumphosphate buffer, pH 7.4) and sonicated (17). (3-Lactamaseactivity was determined spectrophotometrically (18) at 255nm, using 100 uM cephalosporin C as substrate in 10 mMMgCl2/10 mM potassium phosphate buffer, pH 7.4. Specificactivity was expressed as ,mol of substrate hydrolyzed permin at 22°C per mg of total protein (19).

Induction of f-Lactamase Expression in C. freundu and E.coli. Bacteria were grown exponentially in M9CA medium forat least eight generations to an OD420 of 0.8. Subsequently,they were diluted into an equal volume of prewarmedmedium containing twice the final inducer concentration.Samples, 20 or 40 ml, were withdrawn at various times intocentrifuge tubes on ice, containing chloramphenicol (200,g/ml) to inhibit protein synthesis. Relative 83-lactamaseproduction was then determined.

Induction of ,B-Lactamase Expression in Minicells. StrainAA10, obtained from P. Orndorff (Stanford University), is arecA derivative of P678-54 (20) and was used for the minicellexperiments. Minicells from AA10 harboring the approprateplasmids were prepared (21) and finally resuspended to OD600= 1.0 in M9 medium (15) supplemented with 10% methionineassay medium (Difco). Aliquots (200 ,ul) were then preincu-bated at 37°C for 30 min, 40 uCi (1 Ci - 37 GBq) of[35S]methionine (Amersham) was added, and after 2 min thepulse was ended by adding 200 ,ul ofL broth with methionineat 100 ,ug/ml, followed by incubation for an additional 10 min.The minicells were then prepared for electrophoresis asdescribed (22). Equal amounts of trichloroacetic acid-precip-itable radioactivity from the different samples were electro-phoresed on sodium dodecyl sulfate/15% polyacrylamidegels (23). The gels were fixed, stained, treated withEN3HANCE (New England Nuclear), and fluorographed for

Abbreviations: bp, base pair(s); 6-APA, 6-aminopenicillanic acid.

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Proc. NatL Acad. Sci USA 82 (1985) 4621

12-96 hr. For induction, ampicillin was added to a finalconcentration of 1 mg/ml at 0, 2, 5, 10, or 15 min prior to the[35S]methionine pulse.

RESULTSIdentification ofa Gene Coding for a Trans-Acting Regulator

of f-Lactamase Synthesis. E. coli SN03 (ampAl, ampC8,pyrB, recA, rpsL) is a chromosomal /-lactamase hypo-producer (24) and is resistant to ampicillin at no more than 1pg/ml when grown on M9CA medium (Table 1). When theplasmid pNU302 (ref. 4; Fig. 1) carrying an EcoRP fragmentcoding for the ampC ,3-lactamase of C. freundii was used totransform SNO3, the ampicillin resistance increased to 11,ug/ml due to synthesis of 3-lactamase from the plasmid. APst I cutback derivative of pNU302, designated pNU305(Fig. 1), conferred the same ampicillin resistance on SNO3 asdoes the parent plasmid, and expressed the same amount of(-lactamase (Table 1). However, pNU307, a derivative ofpNU305 with a deletion of the Bgl II/BamHIj fragment,could make the transformants resistant to the drug at 30,g/nml. When the pACYC184 (25) derivative pNU311 (Fig.1), carrying the Sal 11/Cla I, region of pNU302, was intro-duced into SN03 together with pNU307 by transformation,the ampicillin resistance was only to 11 pg/ml, and the rateof /3-lactamase synthesis decreased correspondingly (Table1). In a cotransformation experiment using pNU327 (Fig. 1),which lacks the C. freundii DNA insert, and pNU307, thisrepression was not observed. Neither plasmid pNU312,carrying one half of the pNU311 insert (Fig. 1), nor pNU313,carrying the other, had any influence on (-lactamase expres-sion from pNU307.

Table 1. Relative fi-lactamase activity expressed from the clonedampC gene of C. freundii in E. coli SNO3 undernoninducing conditions

Ampicillin Relative P-lactamasePlasmid(s) LD50, ,ug/ml specific activity

None <1 <0.02pNU302 11 0.9pNU305 11 1.0pNU305/pNU311 10 0.9pNU305/pNU312 11 1.1pNU305/pNU313 11 1.0pNU305/pNU327 11 1.0

pNU307 30 2.5pNU307/pNU311 11 1.0pNU307/pNU312 30 2.7pNU307/pNU313 28 2.6pNU307/pNU327 29 2.6

pNU314 30 2.4pNU314/pNU311 11 0.9pNU314/pNU312 30 2.4pNU314/pNU313 30 2.5pNU314/pNU327 30 2.4

pNU337 11 1.0pNU338 10 1.1pNU339 11 0.9

p-Lactamase activity is given as enzyme units per mg of totalprotein relative to pNU305/SNO3. The values given are the mean oftwo or three experiments. The relative ,-lactamase activity ex-pressed in M9CA medium from pNU305/SNO3 is approximately 15times that expressed from C. freundii OS60. Plasmids pNU311,pNU312, pNU313, pNU315, or pNU327 in SNO3 give resultsidentical to those obtained with SNO3 alone.

To more precisely define the regulatory region responsiblefor this pattern of 3-lactamase regulation, we introducedframeshift mutations by inserting a 10-bp DNA adaptor intoeither BamHI site of pNU305 (Fig. 1). Insertion into theBamHI2 site (pNU315), which maps in the ampC gene,resulted in the loss of f3-lactamase expression, whereasinsertion into the BamHIj site (pNU314) resulted in resist-ance to ampicillin at 30 pug/ml and to a 2.5-fold higherproduction of 3-lactamase than from the parent plasmid. Themutation in pNU314 is, however, complemented by pNU311,which reduces the resistance to 11 ,ug/ml (Table 1). Theregion between frdD and ampC of C. freundii, therefore,must code for a trans-acting regulator that represses (-lactamase synthesis by a factor of 2.5 in the absence of3-lactams. We shall tentatively refer to the gene coding forthis proposed regulator as ampR.

Induction in E. coUi of .-L actse Synthesis from pNU305and Its Derivatives. Induction of SN03/pNU305 with 6-amino-penicillanic acid (6-APA, a good inducer) at 2 mg/mlresulted in a more than 11-fold increase in specific (3-lactamase activity (Fig. 2). Expression from the ampRmutant pNU314 could not be induced, whereas in cellscarrying both pNU314 and pNU311 inducibility was re-stored. Since both SN03/pNU305 and SN03/pNU314 +pNU311 could be induced well beyond the constitutive levelexpressed from pNU307 or pNU314 in SNO3, the ampR geneproduct must have a positive effect on 3-lactamase expres-sion in the presence of inducer.To follow the initial phase of induction, E. coli minicells

were pulse labeled with [35S]methionine so that the rate ofampC 3-lactamase synthesis could be monitored (Fig. 3;Materials and Methods). Under these conditions, the rate of/3-lactamase synthesis from pNU305 could be induced 23-foldin 15 min (Fig. 3, lanes A1-A6), with an increase evidentwithin 2 min after the addition of inducer. The relativeexpression of the FrdB, FrdC, and FrdD proteins wasconstant throughout the experiment. The relative rate ofsynthesis from pNU6 (26), a plasmid carrying E. colifrd-ampDNA spanning the DNA corresponding to the C. freundiiinsert in pNU305, was not affected by the induction proce-dure (Fig. 3, lanes D1-D6). Furthermore, no induction wasobtained with minicells harboring the deletion mutantpNU307 (Fig. 3, lanes B1-B6). Inducibility was partiallyrestored in minicells containing pNU307 together withpNU311 (Fig. 3, lanes C1-C6), but 3-lactamase expressioncould not be induced from minicells containing pNU312,pNU313, or pNU327 together with pNU307 (Fig. 4). Experi-ments using the insertion mutant pNU314 gave results similarto those obtained with pNU307 (data not shown).

Identfication of the ampR Gene Product. Minicells carryingpNU311 express a 31-kDa protein (Fig. 4). Inducibility of theC.freundiiampC 3-lactamase was always associated with theexpression of this protein. pNU305 expressed this polypep-tide (too weakly to be seen in Fig. 3), whereas pNU307,pNU312, pNU313, and pNU314 failed to do so (Figs. 3 and4). Hence the structural gene for this protein must be locatedbetween the frdD and ampC genes (Fig. 1) and spanning theBamHI1 site, indicating that this protein is the product of theampR gene. The expression of AmpR itself was not signifi-cantly affected in the induction experiments (data notshown).Mutations Leading to Constitutive Overproduction of C.

freundUi ampC (3-Lactamase Map Outside the ampR-ampCRegion. Mutants of C. freundii OS60 resistant to high levelsof highly ,B-lactamase resistant 8-lactams appeared with afrequency of 10-6 to 10-7. One clone from carbeniciflin at 20ug/ml (OS61) and one from cefotaxime at 5 ,ug/ml (OS62)were passaged twice without selection and then assayed forrelative (-lactamase production. Both mutants producedapproximately 170 times more /3-lactamase per mg of total

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4622 Biochemistry: Lindberg etaLP

EcoRI EcoRI

Pvu II Pst l1 Pvu I1 Pst 12 Pst I 3 Pvu II 2 Pst Pvu II

SailICaISalIClal1 Clal12BamHI Bg III Ia BamHl1 BamHI2 BamHI3 BgIll22 l

.l = = 1 ka _2 1 1 pNU302frd A B C D amp R aMe C

i I 11II I Ix

I I I I I Ix

I I lIl e l ..III I I I I l .?1-A-

e I I I I I _

I I I __u

- z illA? II I

pNU337 pNU338 pNU339

pNU31 1

pNU327

pNU312

pNU313

pBR322

=r: pACYC184

FIG. 1. Restriction map of the plasmids used in this study. All plasmids are derivatives of pNU302. Plasmid pNU305 is a Pst I cutbackderivative of pNU302. To construct pNU314 and pNU315, pNU305 was partially digested with BamHI and ligated to a 50-fold molar excessof nonphosphorylated BamHI adaptors (5'-CCCGGGGATC-3'; Collaborative Research). Linear full-sized molecules were purified from a 0.7%agarose gel and the fragment ends were annealed for 1 hr, first at 370C and then at room temperature. After transformation, plasmid DNA wasisolated, and the position of the insertion was mapped in the full-sized plasmids, aided by the newly introduced Sma I site. An x denotes anadaptor insertion into a BamHI site. This results in a frameshift and thus a disruption of the affected gene. In pNU307 the DNA betweenBgl III and BamHIj is deleted. pNU337, pNU338, and pNU339 carry the Pst 11/Pst I3 region of C. freundii OS60, OS61, and OS62, respectively.pNU311, pNU312, and pNU313 are subclones of the Sal 11/Cia II, BamHIl/Cla II, and Sal I/BamHIl fragments of pNU305, respectively, inpACYC184. pNU327 is a derivative of pACYC184 with a deletion from the Cla I to the Sal I site.

2

E

X /

0=10 ,I/

~~~/

0 10 20 30 40 50 60

FIG. 2. Induction of /3lactamase expression from the cloned C.

freundii ampC gene in E. coil SNO3. Inducer (6-APA at 2mg/nil) was

added and samples were withdrawn after various times and assayed

for specific (3-lactamase activity. Specific activity relative to

uninduced pNU3O5/SNO3 is plotted. *, pNU3O5/SNO3; A,

pNU314/SNO3; and *, pNU314 plus pNU311/SNO3.

protein than the noninduced wild type (OS60). The aminoacid composition of the enzyme purified from these mutantsis identical to that of the wild-type P-lactamase (data notshown). f-Lactamase synthesis from these mutants could notbe induced by 6-APA (2 mg/ml), although the same concen-tration resulted in a 130-fold increase in expression from thewild type in 1 hr (Fig. 5). Thus both OS61 and OS62constitutively overproduce the ampC ,j-lactamase.To characterize these mutants we inserted the EcoRI

fragments carrying thefrd-amp region from the wild type aswell as from the mutants into pACYC184. The Pst I1/Pst 13

(Fig. 1) regions were further subcloned in the same relativeorientation in the Pst I site of pNU78. The plasmids carryingthe Pst 11/Pst 13 region of OS60, OS61, and OS62 were calledpNU337, pNU338, and pNU339, respectively (Fig. 1). Allthese plasmids in SN03 mediated the same ampicillin resist-ance and 3-lactamase production as pNU305 (Table 1). Equalamounts of /3-lactamase were expressed in C. freundii OS60harboring either pNU337, carrying the wild-type frd-ampregion, or pNU338, carrying the same region from the mutantOS61 (0.54 and 0.48 arbitrary units, respectively). Also in C.freundii OS61 the same 83-lactamase level was obtained withboth plasmids (43 and 39 arbitrary units, respectively). Thissuggests that the mutants are altered in gene(s) elsewhere onthe C. freundii genome.By plating E. coli SN03 carrying pNU305 on cefotaxime at

50 ,ug/ml, mutants producing high levels of C. freundii ampCP-lactamase could be obtained. A group of such mutantsconstitutively overproduce the P-lactamase at approximately20 times the SN03/pNU305 level. This phenotype could not

pNU305

pNU314

pNU315

pNU307

. 1 kbp

_=.k I I

1% I I I I I I

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Al A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6

Pre - ArpCAmpC - W W -mp

FrdB - -_

FrdC,D = -_ _

B-Lp - O a* * * -_ B-Lp

Cl C2 C3 C4 C5 C6 Dl D2 D3 D4 D5 D6

Pre - __ Pre

AmpC , La AmpC

~- _ _ _ _,

CAT - - - * ! * X ¢FrdBCA-

FrdC,D = _ FrdC,D

B-Lp _, *3-l-Lp

FIG. 3. Induction of 3-lactamase expression from the clonedampC gene in E. coli minicells. The fluorograms shown are fromminicells containing pNU305 (A lanes), pNU307 (B lanes),pNU307+311 (C lanes), and pNU6 (D lanes). The first lane in eachgroup represents the uninduced preparation whereas the followinglanes show preparations induced 0, 2, 5, 10, and 15 min prior to theinitiation of the pulse. The position of the mature 8-lactamase(AmpC), its precursor (Pre), the chloramphenicol acetyltransferase(CAT) encoded by the pACYC184 derivatives, the P-lipoprotein(3-Lp), and the products ofthefidgenes (FrdB, FrdC, and FrdD) areshown. Also, the AmpR protein (A) and a truncated AmpC protein(*) are indicated. The latter probably results from an artefactualtranslational start within the l3-lactamase gene (data not shown). TheAmpR protein as also expressed from pNU305, but the band is toofaint to be seen in lanes A1-A6.

be transferred to SNO3 with the plasmid. A representative ofthis group, SN0302/pNU305, was cured of the plasmid,which led to loss of ampicillin resistance. The mutantphenotype was regained upon retransformation with pNU305(Table 2), showing that the mutation is located on the E. colichromosome.

Constitutive Overproduction Requires an Intact ampR Gene.When the cured E. coli mutant SN0302 is transformed withpNU307, which carries the C. freundii ampC gene but lacksampR, the enzyme expression is identical to that obtainedwith pNU307 in the parental strain SNO3 (Tables 1 and 2).Transformation with a plasmid carrying only ampR(pNU311) did not result in P-lactamase production. Thus, thephenotype of the E. coli mutant is expressed only in thepresence of intact C. freundii ampC and ampR genes.

DISCUSSION

Compared to the frd-ampC region of E. coli, which has a

noninducible ,B-lactamase, the C. freundii clone contains anadditional 1100 bp ofDNA between the end of thefrd operonand the beginning ofampC (4). We show here that this regionhas a gene, ampR, that encodes a 31-kDa protein. ampR

Al A2 Bi B2 Cl C2 DI D2

Pre PreAmpC= -- _mi _A ___PreC

CAT _ - CAT

FrdC,D= FrdC,D

B-Lp - * M -B-Lp

FIG. 4. Trans complementation of inducibility in E. coliminicells. The induction was done as described in the legend to Fig.3. In all preparations pNU307 carried the cloned ampC gene. Inaddition to this, the compatible plasmids pNU311 (A lanes), pNU312(B lanes), pNU313 (C lanes), or pNU327 (D lanes) were present.Only the noninduced preparations (lanes Al, Bi, etc.) and prepara-tions from cells pulsed after 10-min incubation with ampicillin (A2,B2, etc.) are shown. The labeling of the bands is as in Fig. 3.

mutants expressed the f3-lactamase at 2.5 times the levelexpressed from the wild-type clone. P-Lactamase productionfrom these mutants was no longer inducible. When com-plemented by a compatible plasmid carrying an intact ampRgene, the basal /3-lactamase expression reverted to thewild-type level and inducibility was restored. When plasmidscontaining either of the two halves of the ampR region wereused no complementation was obtained. This strongly sug-gests that the trans effect is due to the ampR gene product andnot the ampR DNA itself. Both with the wild-type clone andin the complementation experiment with ampR the inducedlevel off,-lactamase was well beyond the level obtained byinactivating the ampR gene. Thus the AmpR protein behavesas a trans-active repressor in the absence of inducing /&lactams, whereas in the presence ofl3-lactams it enhances C.freundii ampC 3-lactamase expression.

Experiments with the C. freundii clones in E. coli minicells

U A180

, 160

140

10

100.2

80Z 60

40-

20-

0 10 20 30 40 50 60Induction time (min)

FIG. 5. Induction of P-lactamase expression in C. freundii.Bacteria were grown logarithmically in M9CA medium to OD420 =

0.8 and then diluted into an equal volume of prewarmed mediumcontaining the inducer 6-APA at 4 mg/ml. Samples were withdrawnat the times indicated and the specific 3-lactamase activity wasplotted relative to the noninduced wild type, C. freundii OS60 (o).Also shown are induction curves for OS61 (-) and OS62 (A).

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Table 2. Expression of the cloned C. freundii 3-lactamase in E.coli mutant SN0302 under noninducing conditions

Relative 83-lactamasePlasmid specific activityNone <0.02pNU305 23.3pNU307 2.6pNU311 <0.02

Specific .8-lactamase expression is given relative to SN03/pNU305.

revealed that (8-lactamase induction was apparent within 2min after the addition of the f3-lactam inducer. Inducibilitywas lost in ampR mutants but could be restored bycomplementation with a compatible plasmid carrying thefunctional ampR gene. The synthesis ofAmpR itself was notappreciably influenced by the addition of inducer, demon-strating that the effect is mediated via the AmpR protein itselfand not by a change in AmpR expression. Since the plasmidsare the only genetic material present in the minicells, induc-tion from the C. freundii clone cannot be secondary to theactivation of any E. coli chromosomal gene.At present, we do not know at which level AmpR regulates

ampC f3-lactamase expression. Phenotypically, the arabinosesystem has many features similar to C. freundii ampCregulation. The araC gene product, a 30.5-kDa protein,represses the transcription of the araBAD operon in theabsence of inducer, whereas transcription from the samepromoter is enhanced by AraC when inducer is bound to it(27, 28). Also, in the phosphate uptake and utilization systemin E. coli a protein with both positive and negative effect ongene expression has been described. Here the phoR geneproduct has a dual role, repressing and activating alkalinephosphatase expression in the presence and absence ofphosphate, respectively. This is thought to be mediated viathe effect of the PhoR protein on phoB expression, but theexact mechanisms for this regulation have not so far beenreported (29).Mutants of C. freundii OS60 that constitutively over-

produce the ampC f8-lactamase at 170-180 times theuninduced wild-type level could be obtained at high fre-quency (>10-7). Two such mutants were investigated furtherby cloning their respective frd-ampC regions. These clonesmediated exactly the same level of ampicillin resistance and13-lactamase production in E. coli as the same region clonedfrom the wild type. Both in the wild-type C. freundii and inthe overproducing mutant tested there was no differencebetween f8-lactamase expression from the -mutant and thewild-type clone. These C. freundii mutations must thereforebe located outside of ampR-ampC and represent one orseveral genes involved in P-lactamase induction. If thecorresponding gene product(s) are directly involved in induc-tion they must be expressed also from the E. coli chromo-some, since expression of the cloned C. freundii 3-lactamasecan be induced in E. coli. In E. coli harboring a plasmid withthe C. freundii ampR and ampC genes, chromosomal muta-tions could be isolated that lead to constitutive 20-foldoverproduction of the cloned 83-lactamase. When cells werecured and retransformed with a plasmid carrying ampC butlacking ampR, 83-lactamase expression in the mutant wasidentical to that in the parent. Thus AmpR expression isessential for induction by /3-lactams as well as for constitutiveoverproduction mediated by mutations in one or several

genes present, most probably, in both C. freundii and E. coli.It is tempting to propose that induction is mediated via theeffect of the inducer on some metabolic process-e.g.,peptidoglycan synthesis-occurring in both species. In C.freundii, this process could be sensed by AmpR, leading toincreased /3-lactamase production, whereas expression fromthe noninducible E. coli ampC gene would not be affected.

We thank Monica Persson for excellent technical assistance. Weare grateful to Sven Bergstrom for valuable discussions during theinitial part of this study and to Mell von Gabain and Arne Olsen forexcellent artwork and photography. This work was supported bygrants from the Swedish Natural Sciences Research Council (Dnr3373), the Swedish Medical Research Council (Dnr 5428), and theSwedish Board for Technical Development (Dnr 81-3384).

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