Gene. ! 16 (1992) 75-80 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

75

GENE 06474

A genetic system to study the in vivo role of transcriptional regulators in Escherichia coli (Repressors; activators; RepA protein; plasmids; expression in Escherichia coil; DNA binding proteins)

Jos6 P6rez-Martin and Manuel Espinosa Centro de lnrestigaciones Biologicas. CSIC. E.28006 Madrid (Spain)

Received by M. Salas: 3 September 1991; Revised/Accepted: 6 February/14 February 1992; Received at publishers: 27 February 1992

SUMMARY

A genetic system for studying in vivo the interactions between a transcriptional regulatory protein and its target DNA has been developed for Escherichia coil. It is composed of two compatible plasmids: one high-copy-number promoter-probe vector, and one low-copy-number vector in which the gene encoding the desired protein is cloned under the control of an inducible promoter. The system was successfully tested for its specificity and for dosage analysis by using a combination of the plasmid pLS l-encoded RepA repressor and its target DNA.

INTRODUCTION

Gene expression can be regulated by specific interactions between cis-acting DNA sequences and their recognizing protein counterparts. A full picture of the role of a desired DNA-binding protein is achieved by combination of in vitro with in vivo assays. However, in vivo analyses are mainly focused on studying the physiological activity and biological role of the desired protein, since comparative studies of wt and mutant DNA-binding proteins can be hindered by the requirement of modulation of the intracellular protein levels (Meyer et al., 1980; Klig et al., 1988). To perform such analysis, we have constructed a set of

Correspondence to: Dr. M. Espinosa, Velfizquez 144, CSIC, E-28006 Madrid (Spain) Tel. (341) 5611800: Fax (34 l) 5627518.

Abbreviations: aa, amino acid(s): Ap, ampicillin: pGal,/~-galactosidase; bp, base pair(s): A, deletion: HTH, ~-helix-tum-~-helix: IPTG, isopropylp-D-thiogalactopyranoside: kb. kilobase(s) or 1000 bp: Kin, kanamycin: MCS, multiple cloning site: nt, nucleotide(s): P, promoter: Sin, streptomycin: a resistance/resistant: RBS, ribosome-binding site: Tc, tetracycline: Tn, transposon; wt. wild type: XGal, 5-bromo-4-chloro-3-indolyl/~-D-galactopyranoside: [ ]. denotes plasmid-carrier state: ' (prime), truncated gene at the indicated side.

plasmids which can be used to study the in vivo interaction oftranscription regulatory proteins with their target DNAs in E. coll. To illustrate the feasibility of this type of genetic system, we have used the well characterized transcriptional repressor RepA, encoded by the promiscuous plasmid pLS 1 (Lacks et al., 1986). RepA has 45 aa (dei Solar et al., 1989), it shows the HTH motif present in many DNAbinding proteins (Pabo and Sauer, 1984), and it regulates the pLS 1 repAB operon by exerting a negative control on its own synthesis and on that of the initiator of replication protein RepB (del Solar et al., 1990, del Solar and Espinosa, 1992). High resolution analysis of the contacts between RepA and its target DNA, showed that the protein binds to a 13-bp symmetric element (most likely the operator), within which the -35 region of PA, is located (dei Solar et al., 1990). Upon binding to its target, RepA introduces a strong DNA bend (P6rez-Martin et al., 1989), that hinders the binding of the RNA polymerase to its target (P6rez-Martin and Espinosa, 1991). The rationale of the system is to construct two compatible plasmids, one that should carry the target DNA fused to a reporter gene, and the second that should contain the gene encoding the desired regulatory protein placed under the control of an inducible promoter. In addition, we sought for a gene dosage ratio between target DNA and protein

76 higher than 1, which gives a wide DNA/protein range to perform the assays.

EXPERIMENTAL AND DISCUSSION

(a) Construction of the promoter-probe vector pLSMp The promoter-pr-~be plasmid pLSMp was used as a vector to carry the fused DNA target-reporter gene. Plasmid pLSMp has the following advantages: (i)Fusion to the lacZ gene facilitates a simple, sensitive, and linear (over a wide range) assay (Miller, 1972); (ii) Expression of the reporter gene is proportional to the amount of transcription. This requires the mRNA translation efficiency to be nearly constant, irrespective of the regulatory region to which it is fused. Since the leader region of the P proximal gene of an operon (such as lacZ) may have an unpredictable translation efficiency (Silhavy and Beckwith, 1985), we used the leader and RBS from the trp'-'lacZ fusion of pRZ5605 (Mandecki and Reznikoff, 1982); (ill)The potential effects of upstream translation were minimized oy inserting stop codons in all three reading frames beyond the cloning sites, thus ensuring the uncoupling of any translation initiating in the cloned DNA from the lacZ transcript; (iv)Plasmid pLSMp has multiple copies and several unique cloning sites derived from pUC 19 (Vieira and Messing, 1982). This feature facilitates the fusion of small DNA fragments containing the regulatory site of interest to the reporter gene; and, (v)To avoid read-through transcription from other regions of the vector, the Rho-independent transcription terminator rrnB (Brosius et ai., 1981)was inserted upstream from the MCS. Furthermore, the lacZ and the bla genes were divergently placed. Construction of pLSMp was as follows: a l.l.kb BamHI-Clal fragment from pCON4 (6.5 kb; de Lorenzo et al., 1988) carrying the RBS of trp'-'lacZ fusion was ligated to a 5.4-kb fragment of pMLBI034 (6.25 kb; Silhavy and Beckwith, 1985) which carries the rest of the lacZ gene, the selectable marker (ApR), and the replication region (Fig. IA). The resulting plasmid, termed pEPEI (6.5 kb), was doubly digested with PstI+BamHl, and the large 5.75-kb fragment was ligated to a 1.12-kb Pst I-Bam H 1 fragment from plasmid pBR322rrnB. This latter plasmid was constructed from pBR322 and from pJFII9HE (Furste et al., 1986, see Fig. IA), and carries the rrnB terminator and the pUCI9 MCS. The resulting plasmid, pEPE2 (6.8 kb) was digested with BamHi and ligat~:d together with the small BmnHI fragment ofplasmid pHP45fl (containing the interposon QSm: Prentki and Krisch, 1984). This element carries translation-termination signals in the three possible reading frames, flanking the resistance gene aadA. The resulting plasmid, pEPE3 (8.8 kb) was digest~ with Hindlll to excise the Sm R gene. This diges-

tion leaves behind the fl translation terminators, thus allowing the construction of the promoter-probe plasmid pLSMp (6.9 kb). Cloning of a target promoter in the MCS of pLSMp produces a transcriptional fusion, making this vector suitable to study promoters with nontranslatable RNAs. In addition, the presence of transcription terminators upstream from the MCS and the divergent positioning of the bla and lacZ genes ensures a low (if ally) basal transcription, which may be important for studying low strength promoters.

(b) Construction of the expression vector pLSMtac The vector pLSMtac gives modulated expression of the RepA-regulator protein. To have the repA gene under the control of a expression-controlled promoter, we used a pSC 10 l-based replicon (compatible with the pMB l-based promoter probe vector pLSMp) which carries the P,,,. together with the lacl q gene. It also has the Km R gene from Tn903 and a low number of copies. Construction of pLSMtac is described in Fig. lB. The cassette P,,c-lacl q gene was excised as a 1.6-kb EcoRlNrul fragment from pJF118HE (Furste et al., 1986), and was cloned between the EcoRI and H/ncll sites ofpLG339 (Stocker et al., 1982). This construction exchanges the Tc R gene of pLG339 for the expression cassette. The expression of the desired gene (coding for a regulatory protein) under the control of P,,,,, allows induction with IPTG. Furthermore, since the dosage of the lacl gene is the same as the P,,,,~, it is possible to obtain a step-by-step It,,,. induction. Consequently, the k~cIq/P,,,,, construction in pLSMtac is a good way to modulate the intracellular concentration of the desired protein, which can be done by varying the concentration of IPTG. This is a key feature when performing studies in rive (Amann et al., 1983). An additional advantage of the system pLSMp-pLSMtac, is their differences in copy numbers (about 30 and 5, respectively) which makes a dosage target/protein ratio of about six. A summary of the properties of the system is depicted in Fig. 2. (c) Specificity of the system using the RepA repressor as a model To test the usefulness and the specificity of the pLSMppLSMtac vectors, we used the pLSI plasmid-coded represser RepA because its target and mechanism of repression are well characterized in vitro and in vivo (del Solar et al., 1989; 1990). The repA gene was cloned into pLSMtac to give pLSMtacA (P~rez-Martin and Espinosa, 1991: see legend to Fig. 3). The specificity of the system was tested by cloning in pLSMp three characterized pLS ! promoters. Pr (tet gene: Ballester et al., 1990): P4a (repAB operon: del Solar ct al., 1990; dcl Solar and Espinosa, 1992), and PIs (mall gene: del Solar and Espinosa, 1992). The putative

77

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Fig. i. Construction of pLSMp (A) and of pLSMtac (B). Sources of plasmids are described in sections a and b. Only the relevant restriction sites are marked. Abbreviations: B, 8amHl: C, Clal. E, EcoRl: H, Hindill', Hc. ltincli: N. Nrul: P. Pml: Ps, Pstl: Sm. Sinai. Black boxes represcnt the folImping vcctor rclevant elements: M. MCS: P, tac promoter. T, rn~B transcription terminator. SD. ribosome-binding site and ST, translation stop codons.

78

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Fig. 2. Description of the system. The structures of vectors (not drawn to scale) and a schematic map of the cloning rcgk~n of each are depicted. (A) Promutcr-plobe vector pLSMp: MCS (EcoRh Sa,'l. ~'t', I, Sinai. Bcm~Hh Hi, dill), translation stop codons in all three reading frames, and RBS (from the trp operon). (B) Expression vector pLSMtac: P,.,., and the pUCI3 MCS (Hi, dllh Psth SaIL Hi, clh Xhal. BamHi. Smah Sacl, EcoRi. (C) Idealized scheme of the system. Expression of any regulatory protein (hatched box) cloned under P,,. is repressed until IPTG is added. Then the protein will act on its target DNA (cloned in pLSMp) yielding repression or activation ol' the studied promoter, which can bc monitorized b~/lGal activity.

promoter Pt for the synthesis of the antisense RNA i was cloned (Lacks et al., 1986; del Solar and Espinosa, 1992). E. coil CC118 (Manoil and Beckwith, 1985) was transformed with each of the derivative plasmid pairs (Fig. 3A), and the/IGai activity of the heteroplasmid strains in uninduced and induced cultures was determined. Repression of the/~Gai activity was observed only in the induced cultures of the heteroplasmid strain harboring pLSMtacA and pLSMpAB (Fig. 3A). Since the latter plasmid carries the RepA-operator and P4n, the results confirm that RepA inhibits only its own promoter (del Solar et al., 1990: del Solar and Espinosa, 1992) and demonstrates the specificity of the combination of plasmids constructed by us.

(d) Repression studies in rive The main advantage of our system is that it facilitates the in x,ivo studies of the activity of DNA-binding proteins over sexeral orders of magnitude. This feature is useful to compare the behaxior of a xvt vs. a mutant protein. To demonstrate this. the pLS l-repA gene was mutated b.~ insertion

and removal of the interposon [2Sin (Prentki and Krisch, 198;). The mutated gent should encode a mutant RepA protein (RepAC t) with a predicted intact HTH motif, but with an altered C-end (Fig. 3B). The wt and mutant repA genes wcre cloned separately into p!.S Mtac, and these constructions were transferred to E. coli CC118. Heteroplasmid strains were made by transformation of the above strains with plasmid pLSMpAB (P4n cloned i~to pLSMp). As a control, plasmid pLSMpT (Pt cloned into pLSMp) was used. The strains were induced with increasing amounts of IPTG, and the results (Fig. 3B) showed an IPTG-dependent repression when plasmids harboring the xxt rep.4 genc and the RepA-target xxere present in the same cells. The slope of the repression curve was diminished when the mutant rcT,..I gene :~as anal.xzcd {Fig. 3B). Even though at the highest IPTG concentrations both x~t and mutant gene products shoxved similar levels of repression, at Iox~ amounts of IPTG the RepA mutant exhibited a reduced repression ability, perhaps due to a higher intracellular in.,,tabilit~ or to a defecti~e folding of the protein.

79 - IPTG /

+ IPTG

(e) Conclusions We have developed a system for E. coil which allows the in vivo studies of the interactions between transcriptional regulatory proteins and their target DNAs. Two compatible plasmids, a promoter-probe vector (pLSMp), and a regulated expression vector (pLSMtac) constitute the basis of our system. We have shown the interaction of the plasmid pLS l-encoded repressor RepA with its own target, the specificity of the system, and the capacity to make regulated assays in vivo. Our system has some advantages over other analogous. The 'challenge phage' system (Benson et al., 1986), which relies on the prevention of the lytic growth of phage P22, is not feasible for the analysis of nonfunctional mutants. Other systems use the galK reporter gene (De Boer, 19841, which does not allow an assay as simple as the fiGal employed by us. We believe that the set of vectors described here can help and complement the in vitro studies of DNA-protein interactions.

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et al., 1990). Nevertheless, the differences observed demonstrate that our system is sensitive enough to distinguish between wt and mutant gene products.

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Fig. 3. Tests of the system in E. coil CC118 (araDI39, dlacX74, galE, galK, phoA20, th.y, fl)sE, rpoB, a~gE(am), recA I [A(ara.leu)7697, F']. (A) Specificity of repression by the repA gene product when repA is cloned in pLSMtac (plasmid pLSMtacA). The diagram shows the flGal activity of cultures harboring plasmids in which four pLS ! promoters were cloned in pLSMp. The assay was performed in E. coil CC118[pLSMtacA] cells. The promoter-containing DNA fragments in the different plasmids were cloned in the Sinai site of pLSMp. The resulting recombinant plasmids are: pLSMpl, HgaI-ApaL! fragment from pLS! (bp 140-607; Lacks et al., 1986); pLSMpAB, Mhol-Hinfl fragnlent from pLS5 (bp 2817-352 of pLSl): pLSMpil, Bgll fragment from pLSMIS (P~rez-Martin et al., 19881, and pLSMpT, Hinfl fragment from pLSl (bp 898-2193). Plasmid pLSMtacA was constructed by cloning the ApaLI-Xmnl fragment from pLSl (bp 607-902) in the BamHl site of pLSMtac. The heteroplasmid cultures were not induced (open bars) or induced (filled bars) by induction with 0. I mM IPTG, and the amount of flGal was measured as described (Miller, 19721. Each experiment is the average of four independent determinations. (B) Repression of Pan by the wt and mutant repA gene products at different inducer concentrations. Plasmid pLSMtacACt was constructed by insertion of the Sm interposon at the single St.vl site present within the rep.,I gene. The HTH motif ldel Solar et al., 1989: shaded region|, the C-terminal aa sequence of the wt protein (as determined b~ dei Solar et al.. 19891. and the predicted aa sequence at the C end of the nmtanl protein are indicated at the b,~ttom.

Alternatively, since RepA bends its DNA target (P~rezMartin et al., 1989: P~rez-Martin and Espinosa, 1991), the aa changes at the C-end of the mutant protein could affect its bending ability and, as a consequence, its repression capacity (Zwieb et al.. 19891. Such a possibility has been suggested for the 029 regulato~ p4 gene product (Rojo

ACKNOWLEDG EM ENTS

The technical assistance of M.T. Alda, P. Valiente and R. Galfin, and the art work by A. Hurtado is acknowledged. Research was financed by CICYT, grant BIO910691. REFERENCES Amann, E., Brosius, J. and Ptashne, M.: Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in E. coll. Gene 25 (1983) 167-178. Ballester, S., AIonso, J.C., L6pez, P. and Espinosa, M.: Comparative expression of the pC! 94 cat gene in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coll. Gene 86 (19901 71-79. Benson, N., Sugiono, P., Bass, S., Lynn, V. and Youderian, P.: General selection for specific DNA-binding activities. Genetics !!4 (1984) !-!2. Brosius, J., Dull, TJ., Sleeter, D.D. and Noller, H.F.: Gene organization and primary structure of a ribosomal RNA operon from Escherichia coil. J. Mol. Biol. 148 (19811 107-127. De Boer, H.A.: A versatile plasmid system for the study of prokaryotic transcription signals in Escherichia coll. Gene 30 (1984) 251-255. de Lorenzo, V., Herrero, M. and Neilands, J.B.: pCON4 and pCON5: improved plasmid vectors to study bacterial promoter. FEMS Microbiol. Lett. 50 (1988) 17-23. del Solar. G. and Espinosa. M.: The cop>' number of plasmid pLSI is regulated by two trans-acting plasmid products: the antisense RNA ll and the rcprcssor protein RcpA. Mol. Microbiol. 6 (1992) 83-94. dcl Solar, G., de la Campa, A.G., P~rez-Martin. J., Choli. T. and Espinosa, M.: Purification and characterization of RcpA, a protein involved in the copy number control of plasmid pLS I. Nucleic Acids Res. 17 (1989) 2405-2420.

80 0d So~ar, G., P6rez-Martin, J. and Espinosa, M.: Plasmid pLS l-encocled RepA protein regulates transcription from repdB promoter by binding to a DNA sequence containing a 13-base pair symmetric element. J. Biol. Chem. 265 (1990) 12569-12575. Furste, J.P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. and Lanka, E.: Molecular cloning of the plasmid RP4 primase region in a multi-host range tacP expression vector. Gene 48 ilao,:, a ~ , u u l I19-13i. Klig, L.S., Carey, J. and Yanofsky, C.: Trp reprcssor interactions with the trp, aroH and trpR operators. J. Moi. Biol. 202 (1988) 769-777. Lacks, S.A., Lopez, P., Greenberg, B. and Espinosa, M.: Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLSI. J. Moi. Biol. 192 (1986) 753-765. Mandecki, W. and Reznikoff, W.S.: A lac promoter with a changed distance between - 10 and -35 regions. Nucleic Acid Res. 10 (1982) 903-912. Meyer, B.J., Maurer, R. and Ptashne, M.: Gene regulation at the right operator (On) of bacteriophage lambda. J. Mol. Biol. 139 (1980) 163-194. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, pp. 221-243. Pabo, C.O. and Sauer, R.T.: Protein-DNA recognition. Ann. Rev. Biochem. 53 (1984) 293-321. P6rez-Martin, J. and Espinosa, M.: The RepA represser can act as a

transcriptional activator by inducing DNA bends. EMBO J. 10 (1991) 1375-1382. P6rez-Martin, J., del Solar, G., de la Campa, A.G. and Espinosa, M.: Three regions in the DNA of plasmid pLS l show sequence-directed static bending. Nucleic Acids Res. 16 (1988) 9113-9123. P6rez-Martin, J., del Solar, G., Lurz, R., de la Campa, A.G., Dobrinski, B. and Espinosa, M.: Induced bending of plasmid pLS l DNA by the plasmid-encoded protein RepA. J. Biol. Chem. 264 (1989) 2133421339. Prentki, P. and Krisch, H.M.: In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29 (1984) 303-313. Rojo, F., Zaballos, A. and Salas, M.: Bend induced by the phage ~29 transcriptional activator in the viral late promoter is required for activation. J. Mol. Biol. 211 (1990) 713-725. Siihavy, T.J. and Beckwith, J.R.: Uses of iac fusions for the study of biological problems. Microb. Rev. 49 (1985) 398-418. Stoker, N.G., Fairweather N.F. and Spratt, B.G.: Versatile low-copynumber plasmid vectors for cloning in Escherichia coll. Gene 18 (1982) 335-341. Vieira, J. and Messing, J.: The pUC plasmids, an M 13mp7-dgrived system for insertion mutagenesis and sequencing with synthetic univer. sol primers. Gcn¢ 19 (1982) 259-268. Zwieb, C., Kim, J, and Adhya, S.: DNA bending by negative regulatory proteins: Gai and Lac rcpressors. Genes Dev. 3 (1989) 602-61 I.