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Regulation of the mhp Cluster Responsible for 3-(3-Hydroxyphenyl)propionic Acid Degradation in Escherichia coli

2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês

10.1074/jbc.m303245200

1083-351X

Begoña Torres, Gracia Porras, José L. Garcı́a, Eduardo Dı́az,

Biochemical and biochemical processes

The mhp gene cluster from Escherichia coli constitutes a model system to study bacterial degradation of 3-(3-hydroxyphenyl)propionic acid (3HPP). In this work the regulation of the inducible mhp catabolic genes has been studied by genetic and biochemical approaches. The Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively, show a peculiar arrangement leading to transcripts that are complementary at their 5′-ends. By using Pr-lacZ and Pa-lacZ translational fusions and gel retardation assays, we have shown that the mhpR gene product behaves as a 3HPP-dependent activator of the Pa promoter, being the expression from Pr constitutive and MhpR-independent. DNase I footprinting experiments and mutational analysis mapped an MhpR-protected region, centered at position –58 with respect to the Pa transcription start site, which is indispensable for MhpR binding and in vivo activation of the Pa promoter. Superimposed in the specific MhpR-mediated regulation of the Pa promoter, we have observed a strict catabolite repression control carried out by the cAMP receptor protein (CRP) that allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available and 3HPP is present in the medium. Gel retardation assays revealed that the specific activator, MhpR, is essential for the binding of the second activator, CRP, to the Pa promoter. Such peculiar synergistic transcription activation has not yet been observed in other aromatic catabolic pathways, and the MhpR activator becomes the first member of the IclR family of transcriptional regulators that is indispensable for recruiting CRP to the target promoter. The mhp gene cluster from Escherichia coli constitutes a model system to study bacterial degradation of 3-(3-hydroxyphenyl)propionic acid (3HPP). In this work the regulation of the inducible mhp catabolic genes has been studied by genetic and biochemical approaches. The Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively, show a peculiar arrangement leading to transcripts that are complementary at their 5′-ends. By using Pr-lacZ and Pa-lacZ translational fusions and gel retardation assays, we have shown that the mhpR gene product behaves as a 3HPP-dependent activator of the Pa promoter, being the expression from Pr constitutive and MhpR-independent. DNase I footprinting experiments and mutational analysis mapped an MhpR-protected region, centered at position –58 with respect to the Pa transcription start site, which is indispensable for MhpR binding and in vivo activation of the Pa promoter. Superimposed in the specific MhpR-mediated regulation of the Pa promoter, we have observed a strict catabolite repression control carried out by the cAMP receptor protein (CRP) that allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available and 3HPP is present in the medium. Gel retardation assays revealed that the specific activator, MhpR, is essential for the binding of the second activator, CRP, to the Pa promoter. Such peculiar synergistic transcription activation has not yet been observed in other aromatic catabolic pathways, and the MhpR activator becomes the first member of the IclR family of transcriptional regulators that is indispensable for recruiting CRP to the target promoter. Phenylpropanoic and phenylpropenoic acids and their hydroxylated derivatives are widely distributed in the environment, arising from digestion of aromatic amino acids or as breakdown products of lignin or other plant-derived phenylpropanoids and flavonoids. The bacterial catabolism of these aromatic compounds plays a key role in recycling of such carbon sources in the ecosystem (1Masai E. Harada K. Peng X. Kitayama H. Katayama Y. Fukuda M. Appl. Environ. Microbiol. 2002; 68: 4416-4424Crossref PubMed Scopus (79) Google Scholar, 2Smith M.A. Weaver V.B. Young D.M. Ornston L.N. Appl. Environ. Microbiol. 2003; 69: 524-532Crossref PubMed Scopus (23) Google Scholar). Commonly occurring hydroxycinnamates (caffeate, ferulate, coumarate, etc.) are catabolized via coenzyme A (CoA) derivatives and protocatechuate (2Smith M.A. Weaver V.B. Young D.M. Ornston L.N. Appl. Environ. Microbiol. 2003; 69: 524-532Crossref PubMed Scopus (23) Google Scholar, 3Priefert H. Rabenhorst J. Steinbüchel A. Appl. Microbiol. Biotechnol. 2001; 56: 296-314Crossref PubMed Scopus (466) Google Scholar, 4Venturi V. Zennaro F. Degrassi G. Okeke B.C. Bruschi C.V. Microbiology. 1998; 144: 965-973Crossref PubMed Scopus (97) Google Scholar). On the other hand, aerobic degradation of phenylpropionic acid, 3-(3-hydroxyphenyl)propionic acid (3HPP) 1The abbreviations used are: 3HPP, 3-(3-hydroxyphenyl)propionic acid; CRP, cAMP receptor protein; OR, operator region; LB, Luria Bertani; Km, kanamycin.1The abbreviations used are: 3HPP, 3-(3-hydroxyphenyl)propionic acid; CRP, cAMP receptor protein; OR, operator region; LB, Luria Bertani; Km, kanamycin. and 3-hydroxycinnamic acid usually involves an initial oxygenation step with formation of 2,3-dihydroxyphenylpropionate (2,3-dihydroxyphenylcinnamate) that is further degraded via a meta-cleavage hydrolytic pathway (5Burlingame R. Chapman P.J. J. Bacteriol. 1983; 155: 424-426Crossref PubMed Google Scholar). These pathways have been described in different bacterial genera (Pseudomonas, Arthrobacter, Ralstonia, Acinetobacter, Comamonas, Escherichia, Rhodococcus) (5Burlingame R. Chapman P.J. J. Bacteriol. 1983; 155: 424-426Crossref PubMed Google Scholar, 6Andreoni V. Bestetti G. Appl. Environ. Microbiol. 1986; 52: 930-934Crossref PubMed Google Scholar, 7Coulson C.B. Evans W.C. Chem. Ind. 1959; 17: 543-544Google Scholar, 8Dagley S. Chapman P.J. Gibson D.T. Biochem. J. 1965; 97: 643-650Crossref PubMed Scopus (31) Google Scholar, 9Levy C.C. J. Biol. Chem. 1967; 242: 747-753Abstract Full Text PDF PubMed Google Scholar, 10Strickland S. Massey V. J. Biol. Chem. 1973; 248: 2944-2952Abstract Full Text PDF PubMed Google Scholar, 11Arai H. Yamamoto T. Ohishi T. Shimizu T. Nakata T. Kudo T. Microbiology. 1999; 145: 2813-2820Crossref PubMed Scopus (41) Google Scholar, 12Powell J.A.C. Archer J.A.C. Antonie van Leeuwenhoek. 1998; 74: 175-188Crossref PubMed Scopus (16) Google Scholar, 13Barnes M.R. Duetz W.A. Williams P.A. J. Bacteriol. 1997; 179: 6145-6153Crossref PubMed Google Scholar), but only a few reports about their genetic characterization have been published (11Arai H. Yamamoto T. Ohishi T. Shimizu T. Nakata T. Kudo T. Microbiology. 1999; 145: 2813-2820Crossref PubMed Scopus (41) Google Scholar, 12Powell J.A.C. Archer J.A.C. Antonie van Leeuwenhoek. 1998; 74: 175-188Crossref PubMed Scopus (16) Google Scholar, 13Barnes M.R. Duetz W.A. Williams P.A. J. Bacteriol. 1997; 179: 6145-6153Crossref PubMed Google Scholar, 14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar, 15Díaz E. Ferrández A. García J.L. J. Bacteriol. 1998; 180: 2915-2923Crossref PubMed Google Scholar). The 3HPP (and 3-hydroxycinnamic acid) degradation pathway from Escherichia coli is encoded by the mhp cluster located at min 8.0 of the genome, and it was the first HPP degradation pathway that was described both at the biochemical and genetic levels (5Burlingame R. Chapman P.J. J. Bacteriol. 1983; 155: 424-426Crossref PubMed Google Scholar, 14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar, 16Burlingame R.P. Wyman L. Chapman P.J. J. Bacteriol. 1986; 168: 55-64Crossref PubMed Google Scholar, 17Díaz E. Ferrández A. Prieto M.A. García J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 523-569Crossref PubMed Scopus (274) Google Scholar). The 3HPP pathway is initiated by the MhpA monooxygenase that transforms 3HPP into 2,3-dihydroxyphenylpropionate, which is then converted to succinate, pyruvate, and acetyl-CoA through the action of a meta-cleavage hydrolytic route that involves a dioxygenase (MhpB), hydrolase (MhpC), hydratase (MhpD), aldolase (MhpE), and acetaldehyde dehydrogenase (MhpF) activity (5Burlingame R. Chapman P.J. J. Bacteriol. 1983; 155: 424-426Crossref PubMed Google Scholar, 17Díaz E. Ferrández A. Prieto M.A. García J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 523-569Crossref PubMed Scopus (274) Google Scholar) (Fig. 1). The mhp cluster is arranged as follows: (i) six catabolic genes encoding the initial monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the hydrolytic meta-cleavage enzymes (mhpCDFE); (ii) a gene (mhpT) that encodes a potential transporter; and (iii) a regulatory gene (mhpR) that is adjacent to the catabolic genes but transcribed in the opposite direction (Fig. 1) (14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar). A similar gene arrangement has been observed in Klebsiella pneumoniae (17Díaz E. Ferrández A. Prieto M.A. García J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 523-569Crossref PubMed Scopus (274) Google Scholar). The mhp cluster of Comamonas testosteroni TA441 also resembles that of E. coli (11Arai H. Yamamoto T. Ohishi T. Shimizu T. Nakata T. Kudo T. Microbiology. 1999; 145: 2813-2820Crossref PubMed Scopus (41) Google Scholar). However, the hpp and ohp clusters responsible for the partial catabolism of 3HPP and 2-hydroxyphenylpropionic acid in Rhodococcus globerulus PWD1 (13Barnes M.R. Duetz W.A. Williams P.A. J. Bacteriol. 1997; 179: 6145-6153Crossref PubMed Google Scholar) and Rhodococcus sp. V49 (12Powell J.A.C. Archer J.A.C. Antonie van Leeuwenhoek. 1998; 74: 175-188Crossref PubMed Scopus (16) Google Scholar), respectively, show a different gene organization and low sequence similarity with the mhp clusters of Gram-negative bacteria (17Díaz E. Ferrández A. Prieto M.A. García J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 523-569Crossref PubMed Scopus (274) Google Scholar). Despite a putative regulatory gene that has been identified in all HPP catabolic clusters described so far, there are no reports on the regulation of such clusters, with the exception of the mhpR gene product from E. coli that was shown to be a transcriptional activator of the mhp catabolic genes (Fig. 1) (14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar). Amino acid sequence comparison analyses revealed that the MhpR protein from E. coli belongs to the IclR family of regulatory proteins (14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar, 17Díaz E. Ferrández A. Prieto M.A. García J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 523-569Crossref PubMed Scopus (274) Google Scholar). Because other IclR-type regulators of aromatic catabolic pathways, i.e. PobR and PcaU of the 4-hydroxybenzoate and protocatechuate degradation in Acinetobacter sp. ADP1 (18DiMarco A.A. Averhoff B. Ornston L.N. J. Bacteriol. 1993; 175: 4499-4506Crossref PubMed Google Scholar, 19Gerischer U. Segura A. Ornston L.N. J. Bacteriol. 1998; 180: 1512-1524Crossref PubMed Google Scholar), PcaR of the protocatechuate degradation in Pseudomonas putida (20Romero-Steiner S. Parales R.E. Harwood C.S. Houghton J.E. J. Bacteriol. 1994; 176: 5771-5779Crossref PubMed Google Scholar) and Agrobacterium tume-faciens (21Parke D. FEMS Microbiol. Lett. 1997; 146: 3-12Crossref Scopus (46) Google Scholar), and CatR and PcaR of the catechol and protocatechuate degradation in R. opacus 1CP (22Eulberg D. Schlömann M. Antonie van Leeuwenhoek. 1998; 74: 71-82Crossref PubMed Scopus (28) Google Scholar, 23Eulberg D. Lakner S. Golovleva L.A. Schlömann M. J. Bacteriol. 1998; 180: 1072-1081Crossref PubMed Google Scholar), are controlling ortho-cleavage pathways, the MhpR protein of E. coli becomes the first IclR-type regulator that is controlling the expression of a meta-cleavage pathway for degradation of aromatic compounds. On the other hand, there are no reports on the promoters driving expression of the genes involved in catabolism of HPP. Therefore, the mhp pathway from E. coli constitutes an interesting model system to study regulatory features of the catabolism of HPP in bacteria. In this work, we have used both genetic and biochemical approaches to study for the first time the transcriptional regulation of an HPP degradation pathway. Superimposed in the specific MhpR-dependent regulation of the mhp catabolic genes from E. coli, the cAMP receptor protein (CRP) acts as a mandatory activator that tightly adjusts the expression of the catabolic genes to the overall growth status of the cell. A peculiar synergistic transcription activation of the mhp genes by the specific MhpR activator and the CRP global regulator is described. Bacterial Strains, Plasmids, and Growth Conditions—The E. coli strains and plasmids used in this work are listed in Table I. Unless otherwise stated, bacteria were grown in Luria-Bertani (LB) medium (24Sambrook 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 (30Miller J.H. Experiments in Molecular Genetics. 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 3HPP (Lancaster) was added to the M63 minimal medium. Where appropriate, antibiotics were added at the following concentrations: ampicillin (100 μg/ml), chloramphenicol (35 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)Reference or originStrainDH5αendA1 hsdR17 supE44 thi-1 recA1 gyrA(Nalr) relA1 Δ(argF-lac) U169 deoR φ80dlacΔ(lacZ)M1524Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarS17-1λpirTprSmr recA thi hsdRM + RP4::2-Tc::Mu::Km Tn7 λpir phage lysogen25de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (735) Google ScholarCC118λpirΔ(ara-leu) araD ΔlacX74 galE galK phoA thi-1 rpsE (Spr) rpoB(Rizr) argE(Am) recA1 λpir phage lysogen25de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (735) Google ScholarMC4100araD319 Δ(argF-lac)U169 rpsL150 (Smr) relA1 flbB5301 deoC1 ptsF25 rbsR26Prieto M.A. García J.L. Biochem. Biophys. Res. Commun. 1997; 232: 759-765Crossref PubMed Scopus (34) Google ScholarSBS688MC4100 Δcrp26Prieto M.A. García J.L. Biochem. Biophys. Res. Commun. 1997; 232: 759-765Crossref PubMed Scopus (34) Google ScholarAFMCMC4100 spontaneous rifampicin-resistant mutant (Rifr)27Ferrández A. García J.L. Díaz E. J. Biol. Chem. 2000; 275: 12214-12222Abstract Full Text Full Text PDF PubMed Scopus (77) Google ScholarAFSBSBS688 spontaneous rifampicin-resistant mutant (Rifr)27Ferrández A. García J.L. Díaz E. J. Biol. Chem. 2000; 275: 12214-12222Abstract Full Text Full Text PDF PubMed Scopus (77) Google ScholarAFMCALAFMC with chromosomal insertion mini-Tn5Km Pa-lacZThis workAFMCRLAFMC with chromosomal insertion mini-Tn5Km Pr-lacZThis workAFMCRALAFMC with chromosomal insertion mini-Tn5Km mhpR/Pa-lacZThis workAFSBALAFSB with chromosomal insertion mini-Tn5Km Pa-lacZThis workAFSBRLAFSB with chromosomal insertion mini-Tn5Km Pr-lacZThis workAFSBRALAFSB with chromosomal insertion mini-Tn5Km mhpR/Pa-lacZThis workPlasmidpUC18Apr; high copy number cloning vector24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarpUTmini-Tn5KmApr Kmr, R6KoriV RP4oriT, mini-Tn5Km transposon delivery plasmid25de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (735) Google ScholarpSJ3Apr; 'lacZ promoter probe vector, lacZ fusion flanked by NotI sites28Ferrández A. Miñambres B. García B. Olivera E.R. Luengo J.M. García J.L. Díaz E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (170) Google ScholarpVTRBCmr; low copy number Ptrc-driven cloning vector (pSC101 derivative), polylinker flanked by NotI29Pérez-Martín J. de Lorenzo V. Gene. 1996; 172: 81-86Crossref PubMed Scopus (31) Google ScholarpPARApr; pUC18 containing the mhpR gene under control of the Plac and Pr promoters14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google ScholarpPARΔopApr, pPAR harbouring a PaΔop promoterThis workpPALApr, pPAR harbouring the mhpR gene under control of PlacThis workpBT18Cmr; pVTRB harbouring the mhpR gene under control of the Ptrc promoterThis workpALApr; pSJ3 containing a 0.5-kb DNA fragment to produce the Pa-lacZ fusionThis workpRLApr; pSJ3 containing a 0.5-kb DNA fragment to produce the Pr-lacZ fusionThis workpRALApr; pSJ3 containing a 1.1-kb DNA fragment to produce the mhpR/Pa-lacZ fusionThis workpRALΔopApr; pSJ3 containing a 1.1-kb DNA fragment to produce the mhpR/PaΔop-lacZ fusionThis workpUTALApr Kmr, pUTminiTn5 Km containing the 4.4-kb NotI Pa-lacZ fragment from pALThis workpUTRLApr Kmr, pUTminiTn5 Km containing the 4.4-kb NotI Pr-lacZ fragment from pRLThis workpUTRALApr Kmr, pUTminiTn5 Km containing the 5.0-kb NotI mhpR/Pa-lacZ fragment from pRALThis work Open table in a new tab DNA Manipulations and Sequencing—Plasmid DNA was prepared by the rapid alkaline lysis method (24Sambrook 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 (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or by electroporation (Gene Pulser; Bio-Rad). DNA manipulations and other molecular biology techniques were essentially as described (24Sambrook 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 Gene-Clean (BIO-101, Inc., Vista, CA). Oligonucleotides were synthesized on an Oligo-1000 M nucleotide synthesizer (Beckman Instruments, Inc.). Nucleotide sequences were determined directly from plasmids by using the dideoxy chain termination method (31Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar). Standard protocols of the manufacturer (Applied Biosystems Inc.) were used for AmpliTaq FS DNA polymerase-initiated cycle sequencing reactions with fluorescently labeled dideoxynucleotide terminators. The sequencing reactions were analyzed using an ABI Prism 377 automated DNA sequencer (Applied Biosystems Inc.). Construction of E. coli Strains Harboring Chromosomal Insertions of the Pa-lacZ and Pr-lacZ Translational Fusions—By means of RP4-mediated mobilization, plasmids pUTRAL, pUTAL, and pUTRL (Table I, Fig. 2A), which contain mini-Tn5Km hybrid transposons expressing mhpR/Pa-lacZ, Pa-lacZ, and Pr-lacZ fusions, respectively, were transferred from E. coli S17–1λpir into different rifampicin-resistant E. coli recipient strains, i.e. AFMC and AFSB, through biparental filter mating as described previously (25de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (735) 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, AFMCRAL, AFMCAL, AFMCRL, AFSBRAL, AFSBAL, and AFSBRL, and their relevant genotypes are indicated in Table I. Production of the MhpR Activator—The mhpR gene was expressed from the Plac promoter in the high copy number pPAL plasmid (Table I) and from the Ptrc promoter in the low copy number pBT18 plasmid (Table I). To prepare crude extracts containing the MhpR protein, MhpR+ extracts, E. coli DH5α (pPAL) cells were grown in ampicillin-containing LB medium to an A 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 mm Tris-HCl buffer, pH 7.5, containing 10% glycerol, 2 mm β-mercaptoethanol, and 50 mm KCl prior to disruption by passage through a French press (Aminco Corp.) operated at a pressure of 20,000 p.s.i. 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 MhpR– extracts from E. coli DH5α (pUC18) cells were prepared in a similar manner. Protein concentration was determined by the method of Bradford (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using bovine serum albumin as standard. N-terminal Amino Acid Sequence Determination—The amino-terminal sequence of MhpR was determined by Edman degradation with a 477A automated protein sequencer (Applied Biosystem Inc.). A crude extract of E. coli DH5α (pRL) cells was loaded in a SDS-polyacrylamide gel, and the MhpR(100 amino acids)-LacZ(1016 amino acids) fusion protein encoded by plasmid pRL was directly electroblotted from the gel onto a polyvinylidene difluoride membrane as previously described (33Speicher D.W. Methods. 1994; 6: 262-273Crossref Scopus (34) Google Scholar). Truncation of the MhpR Binding Motif—To delete one half-site of the MhpR binding motif (operator region, termed OR), plasmid pPAR (Table I) was linearized with the DraIII restriction enzyme and then treated with T4 DNA polymerase. The resulting plasmid, pPARΔop, was sequenced, and it was shown to harbor the PaΔop promoter that contains a modified OR region lacking one of its half-sites (9-bp deletion) (Fig. 7A). Synthesis of DNA Fragments Used as Probes—The target DNA fragments used as probes were generated by PCR using plasmid pRAL (Table I, Fig. 2) as template. To prepare the Pa-Pr fragment (274 bp), primers PP6 (5′-CCGTCTGCTCATTGTTCTG-3′, which encodes amino acids 2 to 8 of MhpR and hybridizes with the coding strand of the mhpR gene between nucleotides 831–849 of the mhp gene cluster (Fig. 1) (14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar)), and LAC-57 (5′-CGATTAAGTTGGGTAACGCCAGGG-3′, which hybridizes at 57 nucleotides downstream of the lacZ translational start codon), were used. To prepare the Pa fragment (134 bp), primers PP6 and PA3′(5′-GATTTTTTATTGTGCGCTCAG-3′, which hybridizes to the mhpA coding strand at the transcription start region of Pa, see Fig. 1), were used. To obtain the Pr fragment (156 bp), we have used the PR5′(5′-GCGCACAATAAAAAATCATTTAC-3′, which hybridizes to the mhpR coding strand at the transcription start region of Pr, see Fig. 1) and LAC-57 primers. The mhpR-Pa fragment (590 bp) was PCR-amplified by using the PP4 (5′-GGTCTTGTTCCGGGCAAAAGGC-3′, which hybridizes 438 nucleotides downstream of the ATG start codon of mhpR) and PA3′ primers. Finally, to prepare the PaΔop fragment (125 bp) we have used the PP6 and PA3′ primers and the pRALΔop plasmid (Table I) as template. Mapping Transcription Start Sites—E. coli DH5α (pRL) and DH5α (pRAL) cells were grown in LB medium in the presence of 1 mm 3HPP until the cultures reached an A 600 of about 1.0. Total RNA was isolated using the RNA/DNA Midi kit (Qiagen) according to the instructions of the supplier. Primer extension reactions were carried out with the avian myeloblastosis virus reverse transcriptase as described previously (26Prieto M.A. García J.L. Biochem. Biophys. Res. Commun. 1997; 232: 759-765Crossref PubMed Scopus (34) Google Scholar), using primers LAC-57 and PP6. To determine the length of the primer extension products, sequencing reactions of pRL and pRAL were carried out with the same primers, i.e. LAC-57 and PP6, by using the T7 sequencing kit and [α-32P]dATP (Amersham Biosciences) as indicated by the supplier. Products were analyzed on 6% polyacrylamide-urea gels. The gels were dried onto Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Biosciences). To confirm the start site(s) of the Pr promoter, total RNAs were used in a reverse transcription-PCR experiment. Whereas primers PP4 and PaTATA (5′-CGCTCAGTATAGGAAGGGTG-3′, which hybridizes 9 nucleotides downstream of the major transcription start site of Pr, see Fig. 1) amplified a 578-bp fragment, primers PP4 and Pr10 (5′-TTGTTAAAAACATGTAAATG-3′, which hybridizes 3 nucleotides upstream of the major transcription start site of Pr, see Fig. 1) did not amplify any fragment, which confirms the transcription start site(s) of Pr deduced from primer-extension analyses. Gel Retardation Assays—DNA fragments used as probes were labeled at their 5′-end with phage T4 polynucleotide kinase and [γ-32P]ATP (3000 Ci/mmol) (Amersham Biosciences). The DNA probes were purified by the QIAquick nucleotide removal kit (Quiagen). 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 CRP (kindly provided by A. Kolb) in a 20-μl final volume. After incubation for 30 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 Biosciences). DNase I Footprinting Assays—The Pa-Pr DNA fragment to be used as probe was singly 5′-end-labeled at the Pa non-coding strand by using a labeled primer during the PCR amplification reaction. The LAC-57 primer (50 pmol) was 5′-end-labeled with [γ-32P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase. To perform the PCR reaction, 5 pmol of labeled and 7.5 pmol of unlabeled primers were used. The 5′-end-labeled PCR product was purified using the High Pure PCR product purification kit from Roche Applied Science. 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, 10 mm EDTA, 1 nm DNA probe, 50 mg/ml bovine serum albumin, and cell extract in a 25-μl final volume. This mixture was incubated for 20 min at 30 °C, after which 0.15 units of DNaseI (Amersham Biosciences) (prepared in 10 mm CaCl2, 50 mm MgCl2, 125 mm KCl, and 10 mm Tris-HCl, pH 7.5) was added and the incubation continued at 37 °C for 30 s. The reaction was stopped by the addition of 180 μl of a solution containing 0.4 m sodium acetate, 2.5 mm EDTA, 50 μg of tRNA/ml, and 5 μg of salmon DNA/ml. After phenol-chloroform extraction, DNA fragments were precipitated with ethanol absolute, washed with 70% ethanol, dried, and directly resuspended in 5 ml 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 (34Maxam A Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (8988) 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. β-Galactosidase Assays—β-Galactosidase activities were measured with permeabilized cells as described by Miller (30Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). In vivo Characterization of the Pa and Pr Promoters and Their Specific Regulation—As mentioned above, the mhpR regulatory gene is transcribed in the opposite direction to that of the mhpABCDFE catabolic genes involved in 3HPP degradation in E. coli (Fig. 1). Therefore, the intergenic mhpR-mhpA region should contain the Pr and Pa promoters driving expression of the mhp regulatory and catabolic genes, respectively. Whereas the ATG translational start codon of the first catabolic gene (mhpA) has been identified, two potential start codons of the regulatory mhpR gene were proposed (14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar). To define precisely the intergenic mhpR-mhpA region, we have determined the N-terminal amino acid sequence of MhpR as indicated under "Experimental Procedures." Because the N-terminal sequence of MhpR was shown to be MQNNEQT, the mhp intergenic region spans from nucleotide 850 (ATG of mhpR) to nucleotide 1043 (ATG of mhpA) within the reported sequence of the mhp cluster, and MhpR (277 amino acids) becomes 4 amino acids shorter than previously proposed (14Ferrández A. García J.L. Díaz E. J. Bacteriol. 1997; 179: 2573-2581Crossref PubMed Google Scholar) (Fig. 1). To study the Pa and Pr promoters of the mhp cluster, a 0.5-kb DNA fragment containing the intergenic mhpR-mhpA region was PCR-isolated and ligated, at both orientations, to the lacZ reporter gene of the promoter-probe vector pSJ3 (Table I), generating the translational fusion plasmids, pAL (Pa-lacZ) and pRL (Pr-lacZ) (Fig. 2A). Moreover, to analyze the role of the mhpR regulatory gene on the Pa and Pr promoters, we have engineered plasmid pRAL (mhpR/Pa-lacZ), which harbors the mhpR gene under control of its own promoter in cis, to the Pa-lacZ reporter fusion in a pSJ3 derivative (Fig. 2A), and pPAL (Plac-mhpR), which encodes the mhpR gene under control of the heterologous Plac promoter (Fig. 2B). To avoid the high copy number of the pSJ3 derivatives and, thus, to analyze faithfully the mhp regulatory system, the