From the Department of Molecular Microbiology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain
Received for publication, March 28, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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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 (5, 14, 16, 17). 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 (5, 17) (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) (14). A similar gene arrangement has been observed in Klebsiella pneumoniae (17). The mhp cluster of Comamonas testosteroni TA441 also resembles that of E. coli (11). However, the hpp and ohp clusters responsible for the partial catabolism of 3HPP and 2-hydroxyphenylpropionic acid in Rhodococcus globerulus PWD1 (13) and Rhodococcus sp. V49 (12), respectively, show a different gene organization and low sequence similarity with the mhp clusters of Gram-negative bacteria (17).
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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.
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EXPERIMENTAL PROCEDURES |
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DNA Manipulations and SequencingPlasmid DNA was prepared by the rapid alkaline lysis method (24). Transformation of E. coli was carried out using the RbCl method (24) or by electroporation (Gene Pulser; Bio-Rad). DNA manipulations and other molecular biology techniques were essentially as described (24). 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 (31). 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 FusionsBy 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 S171pir
into different rifampicin-resistant E. coli recipient strains,
i.e. AFMC and AFSB, through biparental filter mating as described
previously (25). 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 ActivatorThe 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 DH5 (pPAL) cells were grown in ampicillin-containing LB
medium to an A600 of about 1. Cell cultures were then
centrifuged (3,000 x 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
-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 x 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 DH5
(pUC18) cells
were prepared in a similar manner. Protein concentration was determined by the
method of Bradford (32) using
bovine serum albumin as standard.
N-terminal Amino Acid Sequence DeterminationThe
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 DH5 (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
(33).
Truncation of the MhpR Binding MotifTo 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, pPARop, was sequenced, and it was
shown to harbor the Pa
op promoter that contains a
modified OR region lacking one of its half-sites (9-bp deletion)
(Fig. 7A).
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Synthesis of DNA Fragments Used as ProbesThe 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 831849 of the mhp gene cluster
(Fig. 1)
(14)), 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 Paop fragment (125 bp) we have used the PP6 and
PA3' primers and the pRAL
op plasmid
(Table I) as template.
Mapping Transcription Start SitesE. coli DH5 (pRL)
and DH5
(pRAL) cells were grown in LB medium in the presence of 1
mM 3HPP until the cultures reached an A600 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 (26),
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
[
-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 AssaysDNA fragments used as probes were
labeled at their 5'-end with phage T4 polynucleotide kinase and
[-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
-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 0.5x 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 AssaysThe 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
[-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
-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 (34)
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.
-Galactosidase Assays
-Galactosidase activities
were measured with permeabilized cells as described by Miller
(30).
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RESULTS AND DISCUSSION |
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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 Pa-lacZ, Pr-lacZ, and mhpR/Pa-lacZ fusions were subcloned as NotI-DNA cassettes within mini-Tn5 vectors, giving rise to plasmids pUTAL, pUTRL, and pUTRAL, respectively (Fig. 2A), which were used to deliver by transposition the corresponding translational fusions into the chromosome of E. coli AFMC (Table I). The presence of a strong T7 phage transcriptional terminator downstream of the lacZ fusions and the orientation of such fusions within the mini-Tn5 elements (Fig. 2A) prevented read-through transcription from nearby promoters after insertion into the chromosome of the resulting E. coli strains, AFMCAL (Pa-lacZ), AFMCRL (Pr-lacZ), and AFMCRAL (mhpR/Pa-lacZ) (Table I).
The -galactosidase assays of permeabilized E. coli AFMCRAL
cells grown in glycerol-containing minimal medium in the presence or in the
absence of 3HPP revealed that Pa activation was only observed in the
presence of 3HPP (Fig.
3A). To check the influence of the MhpR protein on the
expression of the reporter Pa-lacZ and Pr-lacZ fusions,
plasmid pPAL (Plac-mhpR) was introduced in E. coli AFMCAL
and AFMCRL strains, and
-galactosidase assays of permeabilized cells
grown in glycerol-containing minimal medium in the presence or in the absence
of 3HPP were carried out. Whereas expression of the Pa-lacZ fusion
required the presence of the mhpR gene and the 3HPP inducer
(Fig. 3B), the
Pr-lacZ fusion was expressed constitutively both in the presence and
in the absence of MhpR and 3HPP (Fig. 3,
C and D). These data taken together indicate
that MhpR is a 3HPP-dependent activator of the Pa promoter, being the
expression of the mhpR gene constitutive and MhpR-independent. In
this sense, MhpR shows a distinct regulatory feature when compared with other
IclR-type regulators of aromatic catabolic pathways, e.g. PcaR from
P. putida and PobR and PcaU from Acinetobacter sp. ADP1, all
of which act as transcriptional activators of the cognate catabolic genes, but
they behave as repressors of their own expression
(3537).
Moreover, it is known that some IclR-type regulators, such as PcaU, act on the
same promoter both as transcriptional activators, in the presence of the
cognate inducer, and repressors, in the absence of the inducer molecule
(38). However, this dual
regulatory role was not observed with the MhpR activator and the Pa
promoter because no
-galactosidase activity was observed in E.
coli AFMCAL cells that lack the mhpR gene (data not shown).
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To determine the transcription initiation sites in the Pa and
Pr promoters, primer extension analyses were performed with total RNA
isolated from E. coli DH5 cells containing plasmids pRAL and
pRL (Fig. 4). The transcription
initiation site in the Pa promoter was mapped 91 nucleotides upstream
of the ATG translation initiation codon of the mhpA gene, showing
putative 10 (TATACT) and 35 (TTGTAG) boxes typical of
70-dependent promoters
(Fig. 1). A major transcription
initiation site was located in the Pr promoter 107 nucleotides
upstream of the ATG translation initiation codon of the mhpR gene
(Fig. 1). Although a second
transcription initiation site in Pr could be located four nucleotides
downstream of the major initiation site
(Fig. 4), we cannot rule out
that such a start site could correspond to the 5'-end of a processed
transcript that is initiated at the major transcription start site. Two
putative 10 boxes (AATGAT, TGTAAA) and the absence of consensus
35 regions characterize the Pr promoter
(Fig. 1). Interestingly, the
Pr and Pa promoters in the mhp cluster originate
two mRNA transcripts whose first 10 nucleotides are complementary, a peculiar
arrangement that has not been described yet in other aromatic catabolic
clusters and that could imply a new regulatory mechanism that requires further
study.
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Binding of MhpR to the Pa and Pr PromotersTo demonstrate
the interaction of the MhpR regulatory protein with the intergenic
mhpR-mhpA region, cell-free extracts from E. coli DH5
(pPAL) were subjected to gel retardation assays using as probe a 274-bp
fragment carrying the mhp intergenic region (Pa-Pr probe). Whereas
extracts containing MhpR were able to retard the migration of the Pa-Pr probe
in a protein concentration-dependent manner, control extracts prepared from
E. coli DH5
(pUC18) cells did not
(Fig. 5A), which
suggests a specific binding of the MhpR protein to the Pa-Pr probe even in the
absence of the 3HPP inducer. To determine whether MhpR binds to the
Pa and Pr promoters or, on the contrary, binds to only one
of these two promoters, gel retardation assays were carried out using the
120-bp Pa probe (contains the Pa promoter) and the 157-bp Pr probe
(contains the Pr promoter) (Fig.
1). Whereas the pattern of retardation of the Pa probe at
different concentrations of the MhpR protein
(Fig. 5B) was similar
to that observed with the Pa-Pr probe (Fig.
5A), no shifted band was observed with the Pr probe even
when up to 1 µg of MhpR+ extract was added to the retardation
assay (Fig. 5C). These
data reveal that the target site for MhpR binding is the Pa rather
than the Pr promoter. Moreover, because the binding of MhpR to the
Pa-Pr and Pa probes appears to be similar, the interaction of the regulatory
protein with its target DNA does not require the Pr promoter region.
These data taken together are in agreement with the lacZ-reporter
fusion experiments reported above showing that MhpR activates transcription
from Pa but does not cause any significant effect on the Pr
promoter.
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Gel retardation assays were also carried out in the presence of different concentrations of the 3HPP inducer molecule at a concentration of MhpR that did not retard completely the migration of the Pa-Pr probe. As shown in Fig. 5D, increasing concentrations of 3HPP improved retardation of the DNA probe, which indicates that although 3HPP is not indispensable for binding of MhpR to the mhp intergenic region in vitro, this inducer molecule facilitates such interaction. Binding of an activator to the target DNA regardless of the presence or absence of the inducer has been also observed with other regulatory proteins of the IclR family (38) as well as with members of other families of regulatory proteins involved in catabolism of aromatic compounds (39, 40). In all these cases, binding of the inducer to the regulatory protein will modify the interaction of such regulators with the RNA polymerase leading to transcriptional activation at the corresponding promoter (40). Further work needs to be carried out to understand the molecular basis of the 3HPP-mediated induction of the Pa promoter.
Characterization of the MhpR-binding SiteTo identify the MhpR-binding site (operator) within the Pa promoter region, DNase I footprinting experiments were performed using the Pa-Pr probe and increasing concentrations of MhpR. As shown in Fig. 6, the MhpR activator protected an OR region centered at position 58 with respect to the transcription start site in Pa promoter (Fig. 1). The OR sequence corresponds to a 17-bp imperfect palindromic motif, GGTGCACCTGGTGCACA, with its pseudo-dyad axis through the central T base (underlined) that defines two 8-bp half-sites. Analyses of the putative mhp clusters from E. coli O157:H7 (41) and K. pneumoniae (data base of unfinished microbial genomes at the NCBI server: www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi), as well as the mhp cluster from C. testosteroni (11), revealed the existence within the presumed Pa promoters of a 17-bp palindromic region (TGTGCACCTGGTGCACA, the central T is an A in C. testosteroni). This region is almost identical to OR of Pa from E. coli K-12 but carrying a T instead of a G as the first nucleotide of the left half-site and, thus, constituting a perfect palindromic repeat. Therefore, all these data strongly suggest that OR is essential for Pa activity. As observed in Fig. 6, increasing concentrations of total protein present in the extracts caused DNaseI protection in the 35 region of the Pa promoter, which may reflect binding of the RNA polymerase to such a region.
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To confirm the critical role of OR in the MhpR-dependent activation of the
Pa promoter, a modified OR that lacks one of its half-sites was
engineered in a mutant Pa promoter (Paop) in
plasmid pPAR
op (Fig.
7A). Plasmid pPAR
op was then used to substitute
the wild-type Pa promoter in plasmid pRAL by
Pa
op, giving rise to plasmid pRAL
op
(Fig. 7A). The
MhpR-dependent activation of the Pa-lacZ and
Pa
op-lacZ fusions was checked in vivo by
assaying
-galactosidase activity in E. coli AFMC cells
harboring plasmids pRAL and pRAL
op, respectively. As shown in
Fig. 7B, the
activation of the Pa
op-lacZ fusion showed a 10-fold
reduction compared with that observed with the wild-type Pa-lacZ
fusion, thus indicating that deletion of one half-site in OR causes a
significant reduction in the MhpR-dependent Pa activity. The effect
of the half-site deletion of OR on binding of MhpR to the Pa promoter
was also analyzed by using the wild-type Pa and the mutant Pa
op
fragments as probes in gel retardation assays. Whereas increasing
concentrations of MhpR caused retardation of the Pa probe, the Pa
op
probe remained unshifted even at the highest amount of MhpR tested
(Fig. 7C). Therefore,
these data taken together indicate that the 17-bp palindromic OR is the
binding site of the MhpR regulator to activate the Pa promoter.
Other activators of aromatic catabolic pathways that belong to the IclR family, such as PobR and PcaU from Acinetobacter sp. ADP1 and PcaR from P. putida, also recognize 17-bp palindromic operator regions with its pseudo-dyad axis through a central base and whose consensus sequence (TGTTCGATAATCGCACA) (19) resembles that of OR from E. coli and its homologs from K. pneumoniae and C. testosteroni (identical nucleotides are underlined). However, whereas in E. coli, and most probably in K. pneumoniae, the MhpR-binding site is located 50 nucleotides upstream of the transcriptional start site of the corresponding promoter, in Acinetobacter sp. ADP1 the PobR- and PcaU-binding sites are located 72 nucleotides upstream of the transcription start sites of the pobA and pcaI genes, respectively (19), and in P. putida the two adjacent PcaR-binding sites are located 4 nucleotides upstream of the pcaI transcriptional start site (36). The different locations of the operators within the respective regulatory regions have been shown to reflect significant differences in the mechanism of transcriptional activation by the cognate regulatory proteins (36, 38).
CRP Mediates a Superimposed Level of Regulation on the Pa
PromoterIt is known that some aromatic catabolic pathways in
E. coli, such as the paa, hca, hpa, and mao-encoded
pathways responsible for the catabolism of phenylacetate, phenylpropionate,
4-hydroxyphenylacetate, and 2-phenylethylamine, respectively, are repressed by
the presence of glucose in the culture medium
(26,
27,
4245).
To check whether the mhp cluster is also under catabolite repression
control, E. coli AFMCRAL cells were grown in glycerol- or
glucose-containing minimal medium in the presence of 3HPP. Whereas
permeabilized cells showed -galactosidase activity when grown in
glycerol and 3HPP, no activity was observed when the cells were grown in
glucose and 3HPP (Fig.
8A), thus suggesting a repression effect on Pa
activity by glucose. To analyze whether the glucose effect was carried out
directly on the Pa promoter or, on the contrary, it was because of a
repression of the mhpR-encoded transcriptional activator, we tested
the expression of the Pr-lacZ fusion in E. coli AFMCRL
cells. The
-galactosidase levels in E. coli AFMCRL cells grown
in glycerol were only 1.6-fold higher than those of cells growing in glucose
as carbon source (data not shown), suggesting that the glucose effect on
mhp expression is carried out mainly at the level of the Pa
promoter. This observation was confirmed by assaying the Pa-lacZ
expression in E. coli AFMCAL cells containing plasmid pBT18
(Fig. 2B), which
expresses the mhpR gene under the control of Ptrc, a
promoter that is not repressed by glucose. Whereas permeabilized E.
coli AFMCAL (pBT18) cells grown in glycerol and 3HPP showed
-galactosidase activity, no significant activity was detected when cells
were grown in glucose and 3HPP (Fig.
8B). These data taken together reveal that mhp
catabolite repression control is accomplished mainly through the Pa
promoter.
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Because the glucose repression effect is usually mediated by the CRP-cAMP
complex in enteric bacteria
(46,
47), we checked whether CRP
was necessary for mhp expression. To accomplish this, the
mhpR/Pa-lacZ, Pa-lacZ, and Pr-lacZ translational fusions
were integrated into the chromosome of an isogenic CRP
strain, E. coli AFSB. When E. coli AFSBRAL and E.
coli AFSBAL (pBT18) CRP strains
(Table I) were grown either in
glycerol- or glucose-containing minimal medium in the presence of 3HPP, no
-galactosidase activity was observed
(Fig. 8, A and
B). On the contrary, CRP E.
coli AFSBRL cells (Table
I) growing in glycerol-containing medium showed
-galactosidase levels that were only 1.6-fold lower than that of
CRP+ E. coli AFMCRL cells growing in the same medium (data
not shown). Therefore, these data reveal that whereas activity of the
Pr promoter is not strictly dependent on CRP, this global regulator
is essential for Pa activity and accounts for the repressor effect of
glucose on the expression of the mhp catabolic genes. The
CRP-dependent regulation level of the 3HPP catabolic pathway of E.
coli is, then, superimposed to the specific MhpR-dependent regulation; it
allows the expression of the mhp catabolic genes when the preferred
carbon source (glucose) is not available and 3HPP is present in the medium. A
similar dependence on the CRP-cAMP complex was described in E. coli
for the expression of the hpa and paa clusters involved in
4-hydroxyphenylacetate and phenylacetate degradation pathways, respectively
(27,
44). A possible explanation
for this tight CRP-mediated regulation may be because aromatic compounds can
enter the cell by passive diffusion; therefore, the repression of an uptake
mechanism (inducer exclusion) used as an additional control to prevent leakage
in the activity of standard CRP-dependent promoters, such as Plac
(48), cannot be applied to
regulate aromatic catabolic pathways.
To confirm the in vivo experiments reported above, gel retardation
assays were performed with purified CRP in the presence of cAMP and the Pa-Pr
fragment as probe. Interestingly, CRP was not able to bind to the Pa-Pr probe
unless MhpR was also present in the retardation assay, thus generating a
ternary DNA-CRP-MhpR complex (Fig.
8C). A similar pattern of band shifting was observed when
the DNA probe used was the mhpR-Pa fragment that contains the Pa
promoter but lacks the Pr promoter region
(Fig. 8C). On the
contrary, when using the Pr probe that contains the Pr promoter but
lacks the Pa region, no binding of MhpR and/or CRP was observed
(Fig. 8C). Binding of
the CRP-cAMP complex to the Pa promoter but not to the Pr
promoter is, therefore, in agreement with the genetic experiments showing a
CRP-dependent activation of Pa. Sequence analysis of the Pa
promoter revealed the existence of a potential CRP-binding site
(TTCTGCATATTAATTGACATTT) centered at position 95.5 relative to the
transcription start point of Pa
(Fig. 1). However, the left
half-site of such a binding motif poorly matches with the consensus sequence
(AANTGTGANNTNNNTCACANTT) for CRP-binding
(47), which could explain the
lack of interaction of CRP with the Pa promoter and the observation
that MhpR is strictly necessary for binding of CRP to its target DNA. Such
co-dependence upon two transcription activators, that is, the fact that MhpR
is essential for the binding of the second activator (CRP), is a mechanism
widely used in eukaryotes, but few examples have been reported in prokaryotes
(49). MhpR becomes the first
regulator of the IclR family that is reported to be indispensable for
recruiting CRP to the cognate promoter, and no example of such co-dependence
has yet been observed for other regulators of aromatic catabolic pathways. The
A-tract located between the putative CRP-binding site and the MhpR-binding
site (OR) (Fig. 1) may act as
an UP element (50)
facilitating the interaction of the RNA polymerase C-terminal domain
with the promoter and CRP, according to a class I mechanism of CRP-dependent
activation (51). Future
efforts will be directed to understanding the synergy between CRP and MhpR and
the molecular mechanisms governing the transcriptional activation of the
Pa promoter.
The synergistic transcription activation by the CRP-cAMP complex and the MhpR activator allows us to classify the Pa promoter of the mhp cluster as a class III CRP-dependent promoter (51). The Pg promoter of the hpa cluster for 3,4-dihydroxyphenylacetic acid degradation and the Pa and Pz promoters of the paa cluster for phenylacetic acid degradation in E. coli can also be considered class III CRP-dependent promoters because they require both CRP and the integration host factor protein as transcriptional activators (27, 44). However, all these promoters show different architectures, and whereas the Pa promoter of the mhp cluster requires a pathway-specific activator (MhpR), promoters from the hpa and paa clusters are controlled by the cognate pathway-specific HpaR and PaaX repressors, respectively (27, 42, 44). This suggests that the molecular mechanisms of transcriptional regulation of the aromatic catabolic clusters in E. coli are highly diverse, and they constitute a useful model system to unravel complex regulatory circuits that involve specific and overimposed levels of regulation.
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FOOTNOTES |
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Present address: Dept. of Molecular and Cell Biology, Centro Nacional de
Biotecnología, Consejo Superior de Investigaciones Científicas,
28049 Madrid, Spain.
To whom correspondence should be addressed: Dept. of Molecular Microbiology,
Centro de Investigaciones Biológicas, Consejo Superior de
Investigaciones Científicas, Ramiro Maeztu, 9, 28040 Madrid, Spain.
Tel.: 34-91-8373112; Fax: 34-91-5625791; E-mail:
ediaz{at}cib.csic.es.
1 The abbreviations used are: 3HPP, 3-(3-hydroxyphenyl)propionic acid; CRP,
cAMP receptor protein; OR, operator region; LB, Luria Bertani; Km,
kanamycin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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