Portal de Periódicos da CAPES

Transcriptional Regulation of the Divergent paaCatabolic Operons for Phenylacetic Acid Degradation inEscherichia coli

2000; Elsevier BV; Volume: 275; Issue: 16 Linguagem: Inglês

10.1074/jbc.275.16.12214

1083-351X

Abel Ferrández, José L. Garcı́a, Eduardo Dı́az,

RNA and protein synthesis mechanisms

The expression of the divergently transcribedpaaZ and paaABCDEFGHIJK catabolic operons, which are responsible for phenylacetic acid (PA) degradation inEscherichia coli, is driven by the Pz andPa promoters, respectively. To study the transcriptional regulation of the inducible paa catabolic genes, genetic and biochemical approaches were used. Gel retardation assays showing that the PaaX regulator binds specifically to the Pa andPz promoters were complemented with in vivoexperiments that indicated a PaaX-mediated repression effect on the expression of Pa-lacZ and Pz-lacZ reporter fusions. The region within the Pa and Pz promoters that is protected by the PaaX repressor in DNase I footprinting assays contains a conserved 15-base pair imperfect palindromic sequence motif that was shown, through mutational analysis, to be indispensable for PaaX binding and repression. PA-coenzyme A (PA-CoA), but not PA, specifically inhibited binding of PaaX to the target sequences, thus confirming the first intermediate of the pathway as the true inducer and PaaX as the only bacterial regulatory protein described so far that responds to an aryl-CoA compound. Superimposed in the specific PaaX-mediated regulation is transcriptional activation by the cAMP receptor protein and the integration host factor protein. These global regulators may adjust the transcriptional output from Paand Pz promoters to the overall growth status of the cell. The expression of the divergently transcribedpaaZ and paaABCDEFGHIJK catabolic operons, which are responsible for phenylacetic acid (PA) degradation inEscherichia coli, is driven by the Pz andPa promoters, respectively. To study the transcriptional regulation of the inducible paa catabolic genes, genetic and biochemical approaches were used. Gel retardation assays showing that the PaaX regulator binds specifically to the Pa andPz promoters were complemented with in vivoexperiments that indicated a PaaX-mediated repression effect on the expression of Pa-lacZ and Pz-lacZ reporter fusions. The region within the Pa and Pz promoters that is protected by the PaaX repressor in DNase I footprinting assays contains a conserved 15-base pair imperfect palindromic sequence motif that was shown, through mutational analysis, to be indispensable for PaaX binding and repression. PA-coenzyme A (PA-CoA), but not PA, specifically inhibited binding of PaaX to the target sequences, thus confirming the first intermediate of the pathway as the true inducer and PaaX as the only bacterial regulatory protein described so far that responds to an aryl-CoA compound. Superimposed in the specific PaaX-mediated regulation is transcriptional activation by the cAMP receptor protein and the integration host factor protein. These global regulators may adjust the transcriptional output from Paand Pz promoters to the overall growth status of the cell. phenylacetic acid base pair(s) cAMP receptor protein coenzyme A integration host factor left-half operator region phenylacetyl-coenzyme A polymerase chain reaction right-half Phenylacetic acid (PA)1is a central compound to which pollutants, such as styrene andtrans-styrylacetic acid, as well as other aromatic compounds, such as 2-phenylethylamine, phenylacetaldehyde, and several phenylalkanoic acids with an even number of carbon atoms, converge through different peripheral catabolic pathways (1.Olivera E.R. Miñambres B. Garcı́a B. Muñiz C. Moreno M.A. Ferrández A. Dı́az E. Garcı́a J.L. Luengo J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (173) Google Scholar, 2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Aerobic PA catabolism in Pseudomonas putida U and Escherichia coli W has been reported to represent a novel aerobic hybrid pathway whose first step is the activation of PA to phenylacetyl-coenzyme A (PA-CoA) by the action of a PA-CoA ligase (1.Olivera E.R. Miñambres B. Garcı́a B. Muñiz C. Moreno M.A. Ferrández A. Dı́az E. Garcı́a J.L. Luengo J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (173) Google Scholar,2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In E. coli K-12 and E. coli W, thepaa genes responsible for PA catabolism are clustered in the chromosome at min 31.0 according to the E. coli K-12 linkage map (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The 14 paa genes are organized in three transcription units as follows: two divergently transcribed operons,paaZ and paaABCDEFGHIJK, encoding the catabolic genes and whose expression is driven by the Pz andPa promoters, respectively, and the paaXY operon expressing the paaX regulatory gene from the Pxpromoter (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The absence of the paa genes in E. coli W14, an E. coli W mutant strain (3.Ferrández A. Prieto M.A. Garcı́a J.L. Dı́az E. FEBS Lett. 1997; 406: 23-27Crossref PubMed Scopus (52) Google Scholar), leads to a PA− phenotype (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Previous work involving Pa-lacZ translational fusions revealed that the Pa promoter is negatively regulated by thepaaX gene product since the absence of the latter caused a constitutive expression of the reporter fusion (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The PaaX repressor is a 316-amino acid protein that shows 41.4% amino acid sequence identity with its ortholog PhaN (recently renamed as PaaN (4.Garcı́a B. Olivera E.R. Miñambres B. Fernández-Valverde M. Cañedo L.M. Prieto M.A. Garcı́a J.L. Martı́nez M. Luengo J.M. J. Biol. Chem. 1999; 274: 29228-29241Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar)) from the PA degradation pathway of P. putida U (1.Olivera E.R. Miñambres B. Garcı́a B. Muñiz C. Moreno M.A. Ferrández A. Dı́az E. Garcı́a J.L. Luengo J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (173) Google Scholar) and contains a stretch of 25 residues at amino acids 39–64 that shares similarity with the helix-turn-helix motif for DNA recognition and binding of transcriptional repressors from the GntR family such as FadR (5.Raman N. Black P.N. DiRusso C.C. J. Biol. Chem. 1997; 272: 30645-30650Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and GntR (6.Fujita Y. Miwa Y. J. Biol. Chem. 1989; 264: 4201-4206Abstract Full Text PDF PubMed Google Scholar). It is worth mentioning that whereas activator proteins are very common in transcriptional regulation of aromatic catabolic operons, only a few repressors have been described so far in biodegradation of aromatic compounds, namely HpaR (HpcR) for the catabolism of homoprotocatechuic acid in E. coli (7.Prieto M.A. Dı́az E. Garcı́a J.L. J. Bacteriol. 1996; 178: 111-120Crossref PubMed Google Scholar, 8.Roper D.I. Fawcett T. Cooper R.A. Mol. Gen. Genet. 1993; 237: 241-250Crossref PubMed Scopus (58) Google Scholar), CymR for the catabolism of p-cymene in P. putidaF1 (9.Eaton R.W. J. Bacteriol. 1997; 179: 3171-3180Crossref PubMed Google Scholar), and AphS for the catabolism of phenol in Comamonas testosteroni TA441 (10.Arai H. Akahira S. Ohishi T. Kudo T. Mol. Microbiol. 1999; 33: 1132-1140Crossref PubMed Scopus (39) Google Scholar). The repressor effect of PaaX on the Pa promoter in E. coli W14(lacZ −) cells, containing thePa-lacZ fusion into the chromosome and the paaXgene in a plasmid, could not be alleviated by growing the cells in the presence of PA, suggesting that this aromatic compound is not the inducer of the pathway (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). However, the simultaneous expression of genes paaX and paaK, the latter encoding the PA-CoA ligase that catalyzes the activation of PA to PA-CoA, allowed activity of the Pa promoter when the cells were grown in the presence of PA, suggesting that PA-CoA rather than PA is the true inducer of the pathway (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Due to the unusual catabolic and regulatory features mentioned above, the PA biodegradation pathway becomes a very interesting model of an aerobic hybrid route for the catabolism of aromatic compounds. In this work, we have performed both in vivo and in vitro experiments to investigate the regulation of gene expression of the paaZ and paaABCDEFGHIJK catabolic operons from E. coli. Superimposed in the specific PaaX-mediated repression, two global regulators, the cAMP receptor protein (CRP) and the integration host factor protein (IHF), act as activators of the gene expression driven by Pa and Pzpromoters. TheE. coli strains and plasmids used in this work are listed in Table I. The E. coli AF15 strain was obtained by transferring the (argF-lac)U169 deletion of E. coliSH210 to E. coli W14Rif through biparental mating (12.de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (743) Google Scholar) and selecting for a clone resistant to tetracycline and rifampicin. Unless otherwise stated, bacteria were grown in Luria-Bertani (LB) medium (11.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) at 37 °C. Growth in M63 minimal medium (16.Miller J.H. Experiments in Molecular Genetic s. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) was achieved at 30 °C using the corresponding necessary nutritional supplements and 20 mm glycerol or 10 mm glucose as carbon source. When required, 1 mm PA was added to the M63 minimal medium. Where appropriate, antibiotics were added at the following concentrations: ampicillin (100 μg/ml), chloramphenicol (35 μg/ml), tetracycline (10 μg/ml), kanamycin (50 μg/ml), and rifampicin (50 μg/ml).Table IBacteria and plasmids used in this studyStrain or plasmidRelevant genotype and characteristic(s)Ref. or originE. coli K-12 DH5αendA1 hsdR17 supE44 thi-1 recA1 gyrA(Nalr) relA1 Δ(argF-lac)U169 deoRφ80dlacΔ(lacZ)M1511.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar S17–1λpirTpr Smr recA thi hsdR − M + RP4::2-Tc::Mu::Km Tn7 λpir phage lysogen12.de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (743) Google Scholar CC118λpirΔ(ara-leu) araDΔlacX74 galE galK phoA thi-1 rpsE (Spr)rpoB(Rifr) argE(am) recA1λpir phage lysogen12.de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (743) Google Scholar S90CΔ(lac, pro) rpsL(Smr)13.Biek D. Cohen S.N. J. Bacteriol. 1989; 171: 2056-2065Crossref PubMed Google Scholar DPB101S90ChimD451::mini-tet13.Biek D. Cohen S.N. J. Bacteriol. 1989; 171: 2056-2065Crossref PubMed Google Scholar DPB102S90ChimA452::mini-tet13.Biek D. Cohen S.N. J. Bacteriol. 1989; 171: 2056-2065Crossref PubMed Google Scholar MC4100araD319 Δ(argF-lac)U169 rpsL150(Smr) relA1flbB5301 deoC1 ptsF25 rbsR14.Prieto M.A. Garcı́a J.L. Biochem. Biophys. Res. Commun. 1997; 232: 759-765Crossref PubMed Scopus (34) Google Scholar SBS688MC4100 Δcrp14.Prieto M.A. Garcı́a J.L. Biochem. Biophys. Res. Commun. 1997; 232: 759-765Crossref PubMed Scopus (34) Google Scholar SH210Hfr (PO2A), Δ(argF-lac)169,zai-736::Tn10(Tcr)15.Schweizer H.P. Boos W. Mol. Gen. Genet. 1983; 192: 293-294Crossref PubMed Scopus (15) Google Scholar AFMCMC4100 spontaneous rifampicin-resistant mutant (Rifr)This work AFSBSBS688 spontaneous rifampicin-resistant mutant (Rifr)This work S90CRifS90C spontaneous rifampicin-resistant mutant (Rifr)This work DPB101RifDPB101 spontaneous rifampicin-resistant mutant (Rifr)This work DPB102RifDPB102 spontaneous rifampicin-resistant mutant (Rifr)This work S90CPAS90CRif with chromosomal insertion mini-Tn5Km2 Pa-lacZThis work S90CPZS90CRif with chromosomal insertion mini-Tn5Km2Pz-lacZThis work DPB101PADPB101Rif with chromosomal insertion mini-Tn5Km2 Pa-lacZThis work DPB101PZDPB101Rif with chromosomal insertion mini-Tn5Km2 Pz-lacZThis work DPB102PADPB102Rif with chromosomal insertion mini-Tn5Km2 Pa-lacZThis work DPB102PZDPB102Rif with chromosomal insertion mini-Tn5Km2 Pz-lacZThis work AFMCPAAFMC with chromosomal insertion mini-Tn5Km2Pa-lacZThis work AFMCPZAFMC with chromosomal insertion mini-Tn5Km2 Pz-lacZThis work AFSBPAAFSB with chromosomal insertion mini-Tn5Km2Pa-lacZThis work AFSBPZAFSB with chromosomal insertion mini-Tn5Km2 Pz-lacZThis workE. coli W W14W Δpaa3.Ferrández A. Prieto M.A. Garcı́a J.L. Dı́az E. FEBS Lett. 1997; 406: 23-27Crossref PubMed Scopus (52) Google Scholar W14RifW14 spontaneous rifampicin-resistant mutant (Rifr)2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar AF15W14Rif ΔlacThis work AF15PZAF15 with chromosomal insertion mini-Tn5Km2Pz-lacZThis workPlasmids pUTmini-Tn5Km2Apr Kmr; R6KoriV RP4oriT, mini-Tn5 Km2 transposon delivery plasmid12.de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (743) Google Scholar pSJ3Aprr; ′lacZ promoter probe vector,lacZ fusion flanked by NotI sites2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pAFPZApr; pSJ3 containing a 446-bpBbuI-BamHI DNA fragment to produce thePz-lacZ fusion2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pAFPZTApr Kmr; pUTmini-Tn5 Km2 containing the 4.4-kbakb, kilobase pair. NotI Pz-lacZ fragment from pAFPZThis work pAFPA1TApr Kmr; pUTmini-Tn5 Km2 containing the 4.4-kb NotIPa-lacZ fragment2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pCK01Cmr; low copy number cloning vector (pSC101 derivative), polylinker flanked byNotI sites2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pAADCmr; pCK01 containing a 15.5-kb DNA fragment carrying the paa cluster2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pAFK5Apr; high copy number pUC19 derivative containing the paaK gene under Plac2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pUC18Apr; high copy number cloning vector11.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar pAFXApr; pUC18 expressing the paaX gene under Plac2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pAFX2Cmr; pCK01 expressing the paaX gene under Plac2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pAFPA2Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePa-lacZ fusion2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar pAFPA2-M1Apr; pSJ3 containing a 255-bp XbaI-BbuI DNA fragment to produce the PaM1-lacZ fusionThis work pAFPA2-M5Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePaM5-lacZ fusionThis work pAFPA2-M9Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePaM9-lacZ fusionThis work pAFPA2-M16Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePaM16-lacZ fusionThis work pAFPA2-M18Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePaM18-lacZ fusionThis work pAFPA2-M22Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePaM22-lacZ fusionThis work pAFPA2-M25Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePaM25-lacZ fusionThis work pAFPA2-M26Apr; pSJ3 containing a 255-bpXbaI-BbuI DNA fragment to produce thePaM26-lacZ fusionThis worka kb, kilobase pair. Open table in a new tab Plasmid DNA was prepared by the rapid alkaline lysis method (11.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformation of E. coli was carried out using the RbCl method (11.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA manipulations and other molecular biology techniques were essentially as described (11.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA fragments were purified using the Geneclean Kit (Bio 101, Inc.). Oligonucleotides were synthesized on an Oligo-1000M nucleotide synthesizer (Beckman Instruments). Nucleotide sequences were determined directly from plasmids by using the dideoxy chain termination method (17.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar). Standard protocols of the manufacturer for Taq DNA polymerase-initiated cycle sequencing reactions with fluorescently labeled dideoxynucleotide terminators (Applied Biosystems Inc.) were used. The sequencing reactions were analyzed using a model 377 automated DNA sequencer (Applied Biosystems Inc.). Base substitutions in the PaaX binding motif were introduced through recombinant PCR using the plasmid pAAD as template. To generate mutations in the left-half site of the PaaX binding motif, a first PCR reaction was performed using two pairs of primers as follows: PA5–1 (5′-CAATCTCGGAATGCGCATG-3′, which encodes amino acids 38–44 of PaaA and hybridizes with the non-coding strand of thepaaA gene between nucleotides 2885 and 2867 of thepaa gene cluster (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), underlined are the bases that reconstruct a BbuI restriction site) and the degenerated MUT-PA5 (5′-CTTTCATAAAACAATGWKMTTCGTGTTTTTAATT-3′, which hybridizes between nucleotides 2688 and 2721 enclosing the transcription start sites of Pa (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), see also Fig. 4, where K is G or T, M is A or C, and W is A or T); and PAP (5′-GGTCTAGAGTTATCAAAATAGAGTGCG-3′, located between thePz and Pa promoters, nucleotides 2611–2631 of the paa gene cluster (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) (see Fig. 4), where the engineeredXbaI site is underlined) and the degenerated MUT-PA3 (5′-AATTAAAAACACGAAKMWCATTGTTTTATGAAAG-3′, which is complementary to the MUT-PA5 oligonucleotide). The resulting two PCR products were mixed and subjected to a further PCR reaction using PAP and PA5–1 as primers. Similarly, to generate mutations in the right-half site of the PaaX binding motif, a first PCR reaction was performed using two pairs of primers: PA5–1 and the degenerated MUT-PA52 (5′-CATAAAACAATGTGATTCGWSWWTTTAATTAATTCACG-3′, which hybridizes between nucleotides 2692 and 2729 enclosing the transcription start sites ofPa (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), see also Fig. 4; where S means C or G); and PAP and the degenerated MUT-PA32 (5′-CGTGAATTAATTAAAWWSWCGAATCACATTGTTTTATG-3′, which is complementary to the MUT-PA52 oligonucleotide). The resulting two PCR products were mixed and subjected to a further PCR reaction using PAP and PA5–1 as primers. The amplified 272-bp fragments were digested with XbaI and BbuI and cloned as 255-bp fragments into the double-digested XbaI+BbuI pSJ3 promoter probe vector, giving rise to the pAFPA2-M plasmids (Table I), which were further sequenced to confirm the nucleotide sequence of the cloned mutant fragments. By means of RP4-mediated mobilization, plasmids pAFPA1T and pAFPZT, which contain mini-Tn5Km2 hybrid transposons expressing Pa-lacZand Pz-lacZ fusions, respectively, were transferred fromE. coli S17–1λpir into different rifampicin-resistantE. coli recipient strains, i.e. AF15, S90CRif, DPB101Rif, DPB102Rif, AFMC, and AFSB, as described previously (12.de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (743) Google Scholar). Exconjugants containing the lacZ translational fusions stably inserted into the chromosome were selected for the transposon marker, kanamycin, on rifampicin-containing LB medium. The resulting strains, AF15PZ, S90CPA, S90CPZ, DPB101PA, DPB101PZ, DPB102PA, DPB102PZ, AFMCPA, AFMCPZ, AFSBPA, and AFSBPZ, and their relevant genotype are indicated in Table I. The paaX gene was expressed from the Plac promoter in the high copy number pAFX plasmid and in the low copy number pAFX2 plasmid. SDS-polyacrylamide gel electrophoresis analyses of crude lysates fromE. coli cells harboring plasmids pAFX or pAFX2 did not show the presence of an intense band corresponding to the PaaX protein when they were compared with crude lysates from cells harboring the parental plasmids, thus indicating that the paaX gene was not overexpressed in these recombinant plasmids. To prepare crude extracts containing the PaaX protein, PaaX+ extracts, E. coli W14 (pAFX) cells were grown in ampicillin-containing LB medium to anA 600 of about 1. Cell cultures were then centrifuged (3,000 × g, 10 min at 20 °C), and cells were washed and resuspended in 0.05 volumes of 20 mmTris-HCl buffer, pH 7.5, containing 10% glycerol, 2 mmβ-mercaptoethanol, and 50 mm KCl, prior to disruption by passage through a French press (Aminco Corp.) operated at a pressure of 20,000 pounds/square inch. The cell debris was removed by centrifugation at 26,000 × g for 30 min at 4 °C. The clear supernatant fluid was decanted and used as crude cell extract. The PaaX− extracts from E. coli W14 (pUC18) cells were prepared in a similar manner. Protein concentration was determined by the method of Bradford (18.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar) using bovine serum albumin as standard. The target DNA fragments used as probes were generated by PCR using plasmid pAAD as template. To prepare the Pz-Pa fragment (483-bp), primers PZ5 (5′-GGGGTGAATCAAACGGCTACG-3′, which encodes amino acids 18–23 of PaaZ and hybridizes with the non-coding strand of thepaaZ gene between nucleotides 2402 and 2420 of thepaa gene cluster (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar)) and PA5–1 were used. To prepare the Pa fragment (274-bp), primers PAP and PA5–1 were used. To prepare the Pz fragment (242-bp), primers SG-1 (5′-CTGTGACAGATTTCGCACTC-3′, which hybridizes between nucleotides 2644 and 2625 of thepaaZ-paaA intergenic region (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), see also Fig. 4) and PZ5 were used. The 217-bp DNA fragments harboring mutations in the PaaX binding motif were PCR-amplified from the pAFPA2-M series of plasmids (Table I) using primers PAP and PA5–4 (5′-CGGGCATCCAGTCCTGTGGCTCG-3′, which encodes amino acids 18–25 of PaaA and hybridizes with the non-coding strand of the paaA gene between nucleotides 2828 and 2806 (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar)). The PAP and PA5–4 primers were also used to amplify the wild-typePa promoter from the pAFPA2 plasmid. DNA fragments used as probes were labeled at their 5′-end with phage T4 polynucleotide kinase (Amersham Pharmacia Biotech) and [γ-32P]ATP (Amersham Pharmacia Biotech). The reaction mixtures contained 20 mm Tris-HCl, pH 7.5, 10% glycerol, 2 mm β-mercaptoethanol, 50 mm KCl, 0.1 nm DNA probe, 50 μg/ml bovine serum albumin, 50 μg/ml salmon sperm (competitor) DNA, and cell extract or purified IHF or CRP in a 20-μl final volume. Purified IHF and CRP were kindly provided by S. Goodman and A. Kolb, respectively. After incubation for 20 min at 30 °C, mixtures were fractionated by electrophoresis in 4% polyacrylamide gels buffered with 0.5× TBE (45 mm Tris borate, 1 mm EDTA). The gels were dried onto Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Pharmacia Biotech). For quantitative definition of the PaaX-DNA interactions, autoradiograms were scanned in a computing densitometer, and the intensity of the bound and unbound DNA bands was measured using the ImageQuant software (Molecular Dynamics). The apparent dissociation constant (K d(app)) of PaaX for the probe DNA was defined as the amount of total protein per ml of reaction mixture at which half of the labeled DNA remained unbound. DNA fragments to be used as probes were singly 5′-end-labeled by using a labeled primer during the PCR-amplification reaction. The desired primer (50 pmol) was 5′-end-labeled with [γ-32P]ATP using T4 polynucleotide kinase. Unincorporated nucleotides were removed in a G-25 Sephadex column (Amersham Pharmacia Biotech), and the labeled primer was eluted with 100 μl of water. To perform the PCR reaction, 25 pmol of both the labeled and unlabeled primers were used. The 5′-end-labeled PCR product was separated in a 2% agarose gel and purified through the Geneclean DNA purification kit (Bio 101). For DNase I footprinting assays, the reaction mixture contained 20 mm Tris-HCl, pH 7.5, 10% glycerol, 2 mm β-mercaptoethanol, 50 mm KCl, 0.1 nm DNA probe, 50 μg/ml bovine serum albumin, 50 μg/ml salmon sperm (competitor) DNA, and cell extract in a 100-μl final volume. This mixture was incubated for 20 min at 30 °C, after which 2 μl (0.005 units) of DNase I (Sigma) (prepared at 1 ng/μl in 125 mm CaCl2, 50 mm MgCl2) was added, and the incubation was continued for 2 min at 37 °C. After phenol/chloroform extraction, ammonium acetate was added to the clear supernatant to a final concentration of 0.2 m, and DNA fragments were precipitated with ethanol absolute, washed with 70% ethanol, and directly resuspended in 5 μl of 90% (v/v) formamide-loading gel buffer (10 mm Tris-HCl, pH 8.0, 20 mm EDTA, pH 8.0, 0.05% (w/v) bromphenol blue, 0.05% (w/v) xylene cyanol). Samples were then denatured at 95 °C for 2 min and fractionated in a 8% polyacrylamide-urea gel. A + G Maxam and Gilbert reactions (19.Maxam A Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9008) Google Scholar) were carried out with the same fragments and loaded in the gels along with the footprinting samples. The gels were dried onto Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Pharmacia Biotech). β-Galactosidase activities were measured with permeabilized cells as described by Miller (16.Miller J.H. Experiments in Molecular Genetic s. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). We have previously shown that the Pa promoter, which drives the expression of the paaABCDEFGHIJK catabolic operon, is repressed by the PaaX regulator (2.Ferrández A. Miñambres B. Garcı́a B. Olivera E.R. Luengo J.M. Garcı́a J.L. Dı́az E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). To check whether the expression of the divergently transcribed Pz promoter, which drives the expression of the paaZ catabolic gene, was also regulated by PaaX, we have engineered a reporter Pz-lacZ fusion within a mini-Tn5 vector. The resulting construction, pAFPZT, was used to deliver by transposition the Pz-lacZ translational fusion