JOURNAL OF BACTERIOLOGY, Nov. 1985, p. 872-8770021-9193/85/110872-06$02.00/0Copyright © 1985, American Society for Microbiology

Vol. 164, No. 2

Negative Regulation of Adenylate Cyclase Gene (cya) Expression byCyclic AMP-Cyclic AMP Receptor Protein in Escherichia coli:

Studies with cya-lac Protein and Operon Fusion PlasmidsMAKOTO KAWAMUKAI,' JIRO KISHIMOTO,' RYUTARO UTSUMI,' MICHIO HIMENO,' TOHRU KOMANO,1*

AND HIROJI AIBA3

Laboratory ofBiochemistry, Department ofAgricultural Chemistry, Kyoto University, Kyoto 606'; Laboratory ofBiochemistry, Department ofAgricultural Chemistry, Kinki University, Higashiosaka 5772; and Department of Chemistry,

The University of Tsukuba, Sakura-Mura, Ibaraki 305,3 Japan

Received 1 April 1985/Accepted 5 August 1985

We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cyaexpression was examined by measuring the synthesis of 3-galactosidase in Escherichia coli cells containingfused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenouscAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZfused protein as a control system, tet expression was not affected by cAMP. The inhibition of cya expression bycAMP was lso observed in the cya-lac fused operon system, although it was necessary to increase the amountof cAMP or CRP in the cells to detect the effect. The results indicate that cAMP-CRP is a negative regulatorof cya expression at the level of transcription.

Cyclic AMP (cAMP) in E:scherichia coli regulates geneexpression at the transcriptional level, coordinating withcAMP receptor protein (CRP). Many genes in E. coli areunder the control of the cAMP-CRP complex. Catabolite-sensitive operons such as lac, ara, mal, and gal are well-known examples (29). Genes responsible for the metabolismof some amino acids, tnaA, ilvB, and dsdA, are otherexamples (14, 29). Some genes of membrane proteins, ompA(21) and ompF (28), and an operon concerning DNA synthe-sis, deo (29), are also regulated by cAMP-CRP. In addition,there are reports that cAMP regulates many cellular func-tions such as flagellum formation (32), lambdoid infection(6), and cell division (31). Considering the pleiotropic regu-latory roles of cAMP in E. coli, we are interested in themechanism by which the intracellular cAMP level is con-trolled.To understand how the levels of cAMP are controlled, the

regulation of adenylate cyclase activity and expression werestudied. The activity of this enzyme seems to be regulated byinteractions with transport proteins in bacteria (9). An im-portant question about the expression of the adenylatecyclase gene (cya) is the role of cAMP and CRP. Someearlier reports suggested that cAMP-CRP might be a repres-sor for the adenylate cyclase gene (10, 17). Recently, cyawas cloned and its promoter region and the entire genomewere analyzed (4, 5, 16, 24, 25). Subsequently, we demon-strated that the transcription of cya is negatively regulatedby cAMP and CRP both in vitro and in vivo (2). However,cya-lac gene fusion studies by two independent groupsprovided somewhat different results concerning the regula-tion of cya by cAMP-CRP (7, 25). Bankaitis et al. found onlya weak repressive effect ofcAMP on cya expression by usingcya-lac fusion strains (7), whereas Roy et al. failed toobserve any repressive effects ofcAMP on cya expression intheir cya-lac fusion plasmids (25). Curiously, in Salmonellatyphimurium, cya expression was clearly repressed bycAMP in the cya-lac fusion system (15).

* Corresponding author.

To resolve such conflicting results concerning the cyaregulation by cAMP-CRP in E. coli, we examined cyaexpression further by using cya-lac fused protein and fusedoperon plasmids. Our data are completely consistent withthe previous finding that cAMP-CRP acts as a transcriptionalrepressor for cya expression.

MATERIALS AND METHODS

Reagents and enzymes. Ampicillin, tetracycline, ando-nitrophenylgalactoside were obtained from Sigma Chemi-cal Co., St. Louis, Mo., and cAMP was purchased from theKohjin Co., Tokyo, Japan. Restriction endonucleases and S1nuclease were purchased from Bethesda Research Labora-tories, Inc., Gaithersburg, Md. T4 DNA ligase was obtainedfrom Takara Shuzo Co., Ltd., Kyoto, Japan. ['y-32P]ATP waspurchased from the Radiochemical Centre, Amersham, U.K.

Bacterial strains and media. The bacterial strains used inthis study are described in Table 1. MacConkey agar (DifcoLaboratories, Detroit, Mich.) containing 1% lactose ormaltose was used to investigate the fermentation of sugars.LB medium (18) was used for the preparation of plasmidDNAs. M9 medium (20) was used for the assays of ,-galactosidase and 1-lactamase.

Isolation of cya- 4kac strains. Strain MJ1023 (cya- Alac)was constructed from strain MC1061 (Alac) by P1 transduc-tion. P1 lysate was prepared from strain TD5818 (ilv- metf-cya Tetr [TnlO]). TD5818 was constructed from TD1000(cya- metE- Tetr [TnJO]). As the TnlO insertion site is veryclose to cya to be cotransduced by P1 (30), a P1 transductantof MC1061 was first selected for tetracycline resistance andits Cya- phenotype was checked on a MacConkey maltoseagar plate. One of the mutants which conferred a Cya-phenotype was named MJ1023. Likewise, RK8008 wasconstructed from MC4100 by the P1 lysate of TD5818.Enzyme assays. P-Galactosidase activity was determined

by the method of Miller (20). M9 medium supplemented with0.2% glucose and 0.2% Casamino Acids (Difco) was used forthe assay medium. Cell growth was measured spectrophoto-metrically at 610 nm.

872

NEGATIVE REGULATION OF cya EXPRESSION IN E. COLI

TABLE 1. Bacterial strains

Strain Genotype Source

MC1061 araD139 A(ara leu)7697 M. J. CasadabanAlac(IPOZY)X74 galU galKhsr hsm + rpsL

MC4100 araDJ39 AIac(IPOZYA) rpsL thi M. J. CasadabanRK8008 cya ilv Tetr (TnlO); other This work

markers same as MC4100MJ1023 cya ilv Tetr (TnlO); other This work

markers same as MC1061TP2010 xyl Acya argH AIacX74 recA ilv A. Danchin

srl::TnlOTP2139 xyl ilvA argH AIacX74 Acrp A. DanchinTD5818 ilv cya metE Tetr (TnJO) This workTD1000 cya metE Tetr (TnlO) R. Utsumi

P-Lactamase activity was determined by the method ofSawai et al. (26). Bacterial culture (1 ml) was centrifuged andresuspended in 0.5 ml of 0.1 M phosphate buffer (pH 7.0).The cell suspension was preincubated at 30°C for 5 min, 50ptl (1,000 U) of penicillin G was added, and the mixture wasleft for 20 to 30 min at 30°C. The enzyme reaction wasstopped by adding 5 ml of iodine reagent, and the A510 of themixture was measured.

Preparation of plasmids and DNA fragments. PlasmidDNAs were purified by the procedure of Birnboim and Doly(8), followed by centrifugation in CsCl. For small-scaleplasmid preparation, the rapid-isolation method of Davis etal. (12) was used. DNA fragments were extracted fromacrylamide gels by the method of Maxam and Gilbert (19).

Transformation. Transformation was done by the RbClprocedure described previously (18). Transformants wereselected on MacConkey agar plates containing 50 jig ofampicillin per ml.

Nucleotide sequence. The DNA sequence was determinedby the method ofMaxam and Gilbert (19) and by the dideoxychain termination method with bacteriophage M13 (27).

RESULTS

Construction of the cya-lacZ and tet-lacZ protein fusionplasmids. As previously reported, the cya gene is transcribedfrom one major promoter, P2, and two weak promoters, P1and P1' (2). We showed that the transcription from P2 isspecifically inhibited by cAMP-CRP (2). We examined theeffect ofcAMP-CRP on cya expression further by using genefusion techniques. To construct a plasmid carrying a cya-lacZ fused protein, the 529-base-pair (bp) BamHI fragment,which contains the cya P2 region including the first 88codons for cyclase (2), was inserted in the BamHI site ofplasmid pMC1403 constructed by Casadaban et al. (11). Twoorientations of the cya BamHI fragment are possible (Fig. 1).Nucleotide sequence data indicated that the cya readingframe would match with that of the lacZ in one of theseorientations (4). Actually we isolated two types of recombi-nant plasmids, pCL1 and pCL2. MC4100 (Alac) cells con-taining pCL1 were phenotypically Lac', while MC4100carrying pCL2 was Lac-.We also constructed a tet-lacZ fused protein plasmid by

using pMC1403. The 80-bp EcoRI-BamHI fragment ofpBR322, which contains the tet promoter region, was placedbetween the EcoRI and BamHI sites of pMC1403, and theresulting plasmid was named pTL1 (Fig. 1). In plasmidpTL1, the reading frame of the tet gene (22) would not matchthat of lacZ. In fact, MC4100 harboring pTL1 showed a

Lac- phenotype, and so we attempted to put both genes inthe proper reading frame by using Si nuclease. PlasmidpTL1 was digested by BamHI and then by S1 nuclease andreligated. The ligation mixture was used to transform E. coliMC4100 (Alac), and selection was made for red colonies onlactose-MacConkey plates containing ampicillin. One of thered colonies was isolated and named pTL2. We analyzedpTL2 by using restriction endonucleases and found that anunknown fragment was inserted into the BamHI site ofpTL1. To find the structure of pTL2 precisely, we deter-mined the nucleotide sequence between cya and lacZ andfound that a 30-bp segment corresponding to the first part ofthe lacZ region was inserted. pTL2 had the proper readingframe for the lacZ gene, thus producing a tet-lacZ chimericprotein (Fig. 1).

Effects of cAMP on expression of cya-lacZ and tet-lacZprotein fusions. To study the effects of cAMP on expressionof the cya-lacZ fusion, we added 1 or 5 mM cAMP to themedium, in which MJ1023 (cya- Alac) harboring plasmidpCL1 was grown, and then measured ,-galactosidase activ-ity at various times (Fig. 2). When cAMP was added,P-galactosidase activity was dramatically inhibited com-pared with the enzyme activity without cAMP (Fig. 2A).There was no significant difference between the addition of 1

pCL1

paL2

pTL1

cya lac

E~~~~~~~~~~~~~~- b

SB

tet lac

E E

pTL2

TGC CCG TOG M TAC AAC GTC GTG ACT OGGAAAACCCG GCG TTAtot i INSERTION |-bIc

FIG. 1. Structure of cya-lacZ and tet-lacZ protein fusion plas-mids. The 529-bp BamHI fragment carrying the major promoter (P2)of cya was ligated to the BamHI site ofpMC1403. Plasmid pCL1 hadthe cya BamHI fragment in the desired orientation to produce acya-lacZ fusion protein. Plasmid pCL2 had the same fragment in theopposite orientation. The 80-bp EcoRI-BamHI fragment containingthe tet promoter region from pBR322 was introduced into pMC1403that had been cleaved with both EcoRI and BamHI. The resultingplasmid pTL1 could not produce a hybrid ,-galactosidase, since thereading frames of tet and lacZ are different in this plasmid. PlasmidpTL1 was digested with BamHI, treated with S1 nuclease, andreligated to form a tet-lacZ protein fusion plasmid pTL2. Thenucleotide sequences around the junction of tet and lac are shownon the bottom. Symbols: B, BamHI; E, EcoRI.

VOL. 164, 1985 873

874 KAWAMUKAI ET AL.

and 5 mM cAMP. On the other hand, in MJ1023 cellsharboring plasmid pTL2, 3-galactosidase activity was notaffected by the addition of cAMP (Fig. 2B). These resultsshow that cAMP specifically inhibited P-galactosidase activ-ity in the cya-lacZ fused protein and indicate that cAMP is anegative regulator of the expression of cya either at thetranscriptional or at the translational level.To test whether different genetic backgrounds of bacteria

affect the effect of cAMP on cya expression, we assayed,-galactosidase activity in other strains containing the fusionplasmids. The reduction of P-galactosidase activity bycAMP was also observed in strain TP2010 A(cya lac) con-taining pCL1, but not in TP2010 harboring pTL2 (Table 2).We also conducted the same experiment with another strain,RK8008 (cya- Alac), and obtained essentially the sameresult as for MJ1023 and TP2010 (data not shown). Theseresults indicate that changing the host background does notchange the regulatory effect of cAMP on cya expression.To rule out the possibility that cAMP reduced ,B-

galactosidase activity by inhibiting the plasmid replication,we determined the P-lactamase activity for a plasmid copy-number indicator. The addition of cAMP showed ratherstimulatory effects for P-lactamase activity (data not shown).This suggests that the inhibitory effect of cAMP on 1B-galactosidase activity is not because of the reduction ofplasmid copy numbers. When 13-galactosidase activity wascorrected for ,B-lactamase activity, the effect of cAMP be-came stronger (Table 2).

Effects ofcAMP on expression of cya-lac fusion in Cya+ andCrp- backgrounds. IfcAMP negatively regulates the expres-sion of cya, one may expect that the level of ,B-galactosidaseactivity in strain MC1061 (cya+ Alac) containing pCL1would be lower than that in the isogenic strain MJ1023 (cya-Alac). Actually this was shown to be the case (Table 2). Wefound that 1-galactosidase activity in the cya+ backgroundwas almost the same as that in the cya- strain supplementedwith 1 mM cAMP. To find whether CRP is involved inrepressing the expression of the cya-lacZ fused protein, we

100

5ct

A

s4

B

100[50F

0 O Q2 03 OA 0.5 0.6 07 0 OJ 02 03 04 0.5 0.6 0.7OD60 06o

FIG. 2. Effects of cAMP on expression of cya-lacZ and tet-lacZfused proteins in strain MJ1023. Cells harboring pCL1 or pTL2 weregrown in M9 medium supplemented with 0.2% glucose, 0.2%Casamino Acids, and 25 Fg of ampicillin per ml. At an opticaldensity at 610 nm of 0.1 to 0.2, cultures were divided into threeportions. To each portion 0 (0), 1 (0), or 5 mM (A) cAMP wasadded, and the incubation was continued. Samples were withdrawnat regular times and assayed for 1-galactosidase activity as de-scribed in Materials and Methods. The activities in MJ1023(pCL1)(A) and in MJ1023(pTL2) (B) were plotted against the optical densityat 610 nm. Units of 13-galactosidase are 103 x optical density at 420nm/(time x ml).

TABLE 2. Effects of cAMP on expression of cya-lacZ andtet-lacZ protein fusions

Strain(plasmid) Concn of cAMP (mM) aiGal 13-Gal/1-lacactiVltya actiVityb

TP2010(pCL1) 0 50 291 22 105 19 5

TP2010(pTL2) 0 78 211 79 235 85 23

MC1061(pCL1) 0 251 245 27

MJ1023(pCL1) 0 641 285 29

TP2139(PCL1) 0 631 625 63

a Activity of 3-galactosidase (,-gal) is expressed as units. One unit isdefined as 103 x optical density at 420 mm/(time x ml). Each strain wasincubated at 37°C until the optical density at 610 nm was 0.1 to 0.2. Sampleswere divided into three portions, and 0, 1, or 5 mM cAMP was added to eachportion. Each sample was incubated until the optical density at 610 nm was0.5, and 0-galactosidase activity was determined as described in Materials andMethods.

bP-Galactosidase activity corrected for P-lactamase activity, which is aindicator of plasmid copy number, is expressed in arbitrary units. 1B-Lactamase and 3-galactosidase were assayed for the same time. Activity of3-lactamase was assayed as described in Materials and Methods. -, Not

determined.

examined the effect of cAMP on P-galactosidase activity inthe crp- strain TP2139 harboring pCL1. Addition of cAMPhad no effect on ,B-galactosidase activity in TP2139 contain-ing pCL1 (Table 2). This indicates that functional CRP isnecessary for the inhibitory effect of cAMP on cya expres-sion. All these results are compatible with the view thatcAMP-CRP negatively regulates cya expression.

Construction of cya-lac operon fusion plasmids. Since pCL1is a protein fusion plasmid, ,-galactosidase activity in strainsharboring this plasmid is an indicator of both transcriptionaland translational regulations for cya. To clarify whether theeffect of cAMP on cya expression is at the transcriptional orthe translational level, we constructed cya-lac operon fusionplasmids. The 529-bp BamHI fragment, containing the cyaP2 region, was ligated to plasmid pMS437C at the BamHIsite. The resulting plasmid was named pC20L (Fig. 3).Plasmid pMS437C, which is derived from both pMC81 andpMC1403, possesses three stop codons in different readingframes in the trpB region to eliminate all possible transla-tional readthrough into the trpA-lac hybrid gene (K.Shigesada, personal communication). Likewise, the 380-bpEcoRI-BamHI fragment containing the cya PlPl' region wasjoined to pMS437C between the EcoRI and BamHI sites toform plasmid pClOL (Fig. 3). We also constructed theadditional cya-lac operon fusion plasmids pC20LA andpClOLA by joining pHA7 with pC20L and pClOL, respec-tively, at the EcoRI site. Plasmid pHA7 carries the crpstructual gene, transcription of which is under the control ofthe bla promoter (3). Thus cells containing pC20LA orpClOLA overproduce CRP.

Effects ofcAMP on expression of cya-lac operon fusions. Asfor the cya-lac fused protein, the effects of cAMP on

J. BACTERIOL.

NEGATIVE REGULATION OF cya EXPRESSION IN E. COLI

E

E B trD

pC 1 OLcya PI1.1I

pC20L

pCIOLAE H

crp

pC20LAEH

crp

E

Elac

lac

E B trp

cya Pl,l'

cya P2

lac EI -I I~~~~~

lac

L.J

1kbFIG. 3. Structure of cya-lac operon fusion plasmids. Plasmid

pMS437C constructed by K. Shigesada is a derivative of pMC1403and pMC81. The 529-bp BamHI fragment carrying cya P2 wasinserted into the BamHI site of pMS437C to form plasmid pC20L.Likewise, plasmid pClOL was constructed by inserting the 380-bpEcoRI-BamHI fragment carrying cya PlPl' into pMS437C. Plas-mids pC20LA and pClOLA were constructed by ligating pHA7,which carries the crp gene (3), with pC20L and pClOL, respec-

tively, at the EcoRI site. Symbols: E, EcoRI; B, BamHI; S, SmaI;H, HindIII.

the nucleotide sequence of the cloned cya gene and foundthat a possible CRP-binding signal exists in the promoterregions (4, 5). Subsequently, we showed that this CRP-binding sequence is a functional site where cAMP-CRP actsto inhibit cya transcription both in vitro and in vivo (2).Although gene fusion techniques are good for monitoring

regulatory signals affecting the expression of genes at thelevels of transcription and translation, there have beenconfusing situations for cya-lac fusions (7, 25). Two groupshave investigated the regulation of cya expression by con-structing cya-lac fusions (7, 25). Bankaitis and Bassfordconstructed lysogens that carry the cya-lac operon orfused proteins by inserting Mu dl(Ap lac) into the cya locuson the chromosome and then replacing it by (7). They usedthese fusion strains to study the effects ofcAMP and CRP oncya expression. They found that the transcription of cya ismoderately repressed by cAMP and that functional CRP isrequired for this effect. However, they concluded that theobserved regulatory effects by cAMP were not physiologi-cally significant, since those effects were very weak. On theother hand, Roy et al. constructed cya-lac operon andprotein fusion plasmids in vitro (25). They examined the roleof cAMP on the cya expression by introducing these fusionplasmids into various strains. They observed no repressiveeffects of cAMP at the transcriptional or translational level.For this report, we have constructed cya-lac protein and

operon fusions in vitro by using pMC1403 and pMS437C,respectively, as plasmid vectors. By measuring the 13-

galactosidase activity of lac- cya- strains containing eachfusion plasmid in the presence and absence of added cAMP,we investigated the regulatory roles ofcAMP on cya expres-sion. Our results showed that ,B-galactosidase activity underthe control of a cya P2 promoter is clearly inhibited by theaddition of cAMP and that the functional crp gene is neces-sary for this effect. Since the repressive effect was observedboth in protein and operon fusions, cAMP-CRP should exertits regulatory function at the transcriptional level. Our fusionstudies are therefore completely consistent with the previousreport (2) that directly showed the transcriptional repressionof cya by cAMP-CRP.The apparent differences between the results of Roy et al.

expression of cya-lac operon fusions was investigated.Strain TP2010 harboring each operon fusion plasmid wascultured in M9 medium either without or with cAMP (1 or 5mM), and the 3-galactosidase activity of cells was measuredat various times. In cells containing pC20L, we observed aninhibition of 1-galactosidase activity with 5 mM but not 1mM cAMP. However, for TP2010 containing pC20LA, astrong repressive effect was observed even with 1 mMcAMP (Table 3). These results indicate that an increasedamount of the cAMP-CRP complex is required for a clearinhibition of the expression of cya-lac operon fusion. Wealso examined the effect of cAMP on the expression of cyaPlPl'-lac operon fusion by using TP2010 containing eitherpClOL or pClOLA. cAMP did not significantly affect -

galactosidase activity in those cells (Table 3).

DISCUSSION

Botsford and Drexler proposed a model of negative con-trol function for the cAMP-CRP complex in regulating cyaexpression at the transcriptional level (10). This model isprimarily based on the observation that crp mutants over-produce cAMP (13, 23). Recently we and others determined

TABLE 3. Effects of cAMP on expression of cya-lac operonfusions

Strain(plasmid) Concn of cAMP atGal 1-Gac/it-lacactiVitya activityb

TP2010(pC20L) 0 380 1121 380 1115 275 52

TP2010(pC20LA) 0 230 1451 180 515 105 25

TP2010(pClOL) 0 120 331 125 325 128 28

TP2010(pClOLA) 0 80 391 93 385 105 31

a 1-Gal, 1-galactosidase.b 13-Galactosidase activity corrected for ,B-lactamase activity (arbitrary

units). Assay conditions are the same as described in Table 2.

-

VOL. 164, 1985 875

876 KAWAMUKAI ET AL.

(25) and our results remain to be explained. First, a proteinfusion plasmid pCL1, with which we showed a strongrepressive effect of cAMP on cya expression, seems to beidentical with pDIA1864 constructed by Roy et al. (25).Unfortunately, however, they gave no experimental dataabout the effect of cAMP on pDIA1876. We conducted thesame experiments as for pCL1 by introducing pDIA1864 intolac- cya- strains and observed a clear inhibitory effect ofcAMP on P-galactosidase activity (data not shown).

Second, structural differences between our operon fusionplasmids and those of Roy et al. (25) seem to be important ingiving inconsistent results. Plasmid pC20L carrying a cyaP2-lac operon fusion is similar to but not identical withpDIA1873 constructed by Roy et al. The former is derivedfrom pMS437C, which is improved as an operon fusionvector by inserting three stop codons to eliminate possibletranslational readthrough into the trpA-lacZ region from theupstream side. Another difference between the two plasmidsis that pC20L carries only a P2 promoter, whereaspDIA1873 contains P1, P1', and P2 promoters. It is notewor-thy that pDIA1873 was toxic to cells in the presence ofcAMP (25). This might be due to the properties of pDIA1873itself, since we observed no growth problem with cellscontaining pC20L in the presence of cAMP.

Third, 1 mM cAMP, which Roy et al. (25) used in theirexperiment, may not be enough to detect the effect of thisnucleotide on the cya-lac fused operon. In fact 1 mM cAMPcaused only a slight inhibitory effect on ,-galactosidaseactivity even for pC20L. To detect a clear effect, it wasnecessary to add more cAMP or to increase CRP concentra-tion or both by joining the crp gene in the same plasmid. It isimportant to point out that the sensitivity of cAMP-CRP tothe fused operon pC20L is lower than that to the fusedprotein pCL1. This might reflect different DNA conforma-tions around the CRP-binding site between the two plasmids.Alternatively, a higher sensitivity in pCL1 could be ex-plained by assuming that cya translation is also regulated bycAMP-CRP. The third possibility is that some artificialtranscription from the upstream promoter that is generatedin the construction may reflect this discrepancy. Experi-ments to test these possibilities are in progress.

ACKNOWLEDGMENTS

We thank K. Shigesada and M. Imai, Institute for Virus Research,Kyoto, Japan, for their advice and supplying plasmid pMS437C. Wealso thank A. Roy and A. Danchin, Institut de BiologiePhysico-Chimique, Paris, France, for supplying strains TP2010 andTP2139.

LITERATURE CITED

1. Aiba, H. 1983. Autoregulation of the Escherichia coli crp gene:CRP is a transcriptional repressor for its own gene. Cell32:141-149.

2. Aiba, H. 1985. Transcription of the Escherichia coli adenylatecyclase gene is negatively regulated by cAMP-cAMP receptorprotein, J. Biol. Chem. 260:3063-3070.

3. Aiba, H., S. Fujimoto, and N. Ozaki. 1982. Molecular cloningand nucleotide sequencing of the gene for E. coli cAMP receptorprotein. Nucleic Acids Res. 10:1345-1361.

4. Aiba, H., M. Kawamukai, and A. Ishihama. 1983. Cloning andpromoter analysis of the Escherichia coli adenylate cyclasegene. Nucleic Acids Res. 11:3451-3465.

5. Aiba, H., M. Kazuyasu, M. Tanaka, T. Ooi, A. Roy, and A.Danchin. 1984. The complete nucleotide sequence of the ade-nylate cyclase gene of Escherichia coli. Nucleic Acids Res.

12:9427-9440.6. Alderman, E. M., S. S. Dills, T. Melton, and W. J. Dobrogosz.

1979. Cyclic adenosine 3',5'-monophosphate regulation of thebacteriophage T6/colicin K receptor in Escherichia coli. J.Bacteriol. 140:369-376.

7. Bankaitis, V. A., and P. J. Bassford, Jr. 1982. Regulation ofadenylate cyclase synthesis in Escherichia coli: studies withcya-lac operon and protein fusion strains. J. Bacteriol. 151:1346-1357.

8. Birmboim, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

9. Botsford, J. L. 1981. Cyclic nucleotides in procaryotes. Micro-biol. Rev. 45:620-642.

10. Botsford, J. L., and M. Drexler. 1978. The cyclic 3',5'-adenosinemonophosphate receptor protein and regulation of 3',5'-adenosine monophosphate synthesis in Escherichia coli. Mol.Gen. Genet. 165:47-56.

11. Casadaban, M. J., J. Chou, and S. N. Cohen. 1980. In vitro genefusions that join an enzymatically active ,3-galactosidase seg-ment to amino-terminal fragments of exogenous proteins: Esch-erichia coli plasmid vectors for the detection and cloning oftranslational initiation signals. J. Bacteriol. 143:971-980.

12. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advancedbacterial genetics, p. 120-121. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

13. Fraser, A. D. E., and H. Yamazaki. 1978. Determination of therates of synthesis and degradation of adenosine 3',5'-cyclicmonophosphate in Escherichia coli K-12 CRP- and CRP+strains. Can. J. Biochem. 56:849-852.

14. Friden, P., T. Newman, and M. Freundlich. 1982. Nucleotidesequence of the ilvB promoter-regulatory region: a biosyntheticoperon controlled by attenuation and cyclic AMP. Proc. Natl.Acad. Sci. USA 79:6156-6160.

15. Jovanovich, S. B. 1985. Regulation of a cya-lac fusion by cyclicAMP in Salmonella typhimurium. J. Bacteriol. 161:641-649.

16. Koop, A. H., M. Hartley, and S. Bourgeois. 1984. Analysis of thecya locus of Escherichia coli. Gene 28:133-146.

17. Majerfeld, I. H., D. Miler, E. Spitz, and H. V. Rickenberg. 1981.Regulation of the synthesis of adenylate cyclase in Escherichiacoli by the cAMP-cAMP receptor protein complex. Mol. Gen.Genet. 181:470475.

18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

19. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeledDNA with base-specific chemical cleavages. Methods Enzymol.65:499-560.

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

21. Mowva, R. N., P. Green, K. Nakamura, and M. Inouye. 1981.Interaction of cAMP receptor protein with the ompA gene. Agene for a major outer membrane protein of Escherichia coli.FEBS Lett. 128:186-190.

22. Peden, K. W. C. 1983. Revised sequence of the tetracycline-resistance gene of pBR322. Gene 22:277-280.

23. Potter, K., G. Chaloners-Larsson, and H. Yamazaki. 1974.Abnormally high rates of cAMP excretion from an E. colimutant deficient in CRP. Biochem. Biophys. Res. Commun. 57:379-385.

24. Roy, A., and A. Danchin. 1982. The cya locus of Escherichia coliK-12: organization and gene products. Mol. Gen. Genet.188:465-471.

25. Roy, A., C. Haziza, and A. Danchin. 1983. Regulation ofadenylate cyclase synthesis in Escherichia coli: nucleotidesequence of the control region. EMBO J. 2:791-797.

26. Sawai, T., I. Takahashi, and S. Yamagishi. 1978. Iodometricassay method for beta-lactamase with various beta-lactam anti-biotics as substrates. Antimicrob. Agents Chemother. 13:910-913.

27. Schreier, P. H., and R. Cortese. 1979. A fast and simple methodfor sequencing DNA cloned in the single-stranded bacterio-phage M13. J. Mol. Biol. 129:169-172.

J. BACTERIOL.

NEGATIVE REGULATION OF cya EXPRESSION IN E. COLI

28. Scott, N. W., and C. R. Harwood. 1980. Studies on the influenceof the cyclic AMP system on major outer membrane proteins ofEscherichia coli K-12. FEMS Microbiol. Lett. 9:95-98.

29. Ullmann, A., snd A. Danchin. 1983. Role of cyclic AMP inbacteria. Adv. Cyclic Nucleotide Res. 15:1-53.

30. Utsui, R. 1984. A simple transductional method for construc-tion of adenylate cyclase or cAMP receptor protein-deficientmutants of Escherichia coli K-12. Agnic. Biol. Chem. 48:

2129-2130.31. Utsumi, R., Y. Nakamoto, M. Kawamukai, M. Himeno, and T.

Komano. 1982. Involvement of cyclic AMP and its receptorprotein in filamentation of an Escherichia coli fic mutant. J.Bacteriol. 151:807-812.

32. Yokota, T., and J. S. Gots. 1970. Requirement of adenosine3',5'-cyclic phosphate for flagella formation in Escherichia coliand Salmonella typhimurium. J. Bacteriol. 103:513-516.

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