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INTRODUCTION |
The TOL plasmid pWW0 of Pseudomonas putida specifies a
meta-cleavage pathway for the oxidative catabolism of
benzoate and toluates. Genes encoding the TOL meta-cleavage
pathway are grouped into a single operon, the expression of which is
positively regulated at the level of transcription by the
xylS gene product, which is activated by benzoate effectors
(1-4). Stimulation of transcription from the Pm promoter requires a
DNA sequence extending to about 80 bp1 upstream of the
transcription initiation point (5-7). On the basis of genetic data,
two regions can be distinguished in the architecture of the Pm
promoter: the XylS interaction region, which extends from about
40 to
80 bp (Fig. 1); and the downstream RNA polymerase recognition region,
which exhibits atypical
35 and
10 DNA sequences (Fig. 1).
Transcription from the Pm promoter in the early exponential growth
phase is mediated by RNA polymerase with
32, and later
with
S, although regardless of the growth phase,
expression from Pm remains dependent on XylS, and the transcription
initiation point is the same (8, 9).
Kessler et al. (6) proposed that the XylS binding region in
Pm was organized as two homologous 15-bp tandemly imperfect directly
repeated motifs (5'-TGCAAPuAAPyGGNTA-3'). The distal one with respect
to the RNA polymerase region extends from
70 to
56, and the
proximal one extends from
49 to
35 (Fig. 1). Gallegos et
al. (5) also studied the organization of the XylS binding sites in
the Pm promoter and suggested that sequences shorter than those
proposed by Kessler et al. (6) might suffice for XylS
activation of transcription. These workers found that promoters that
had been deleted up to
60 could be activated by constitutive XylS
mutants (but not by the wild-type regulator) and that extension of the
deletion to
51 prevented transcription. Gallegos et al.
(5) proposed that the XylS binding site is represented by the motif
T(C/A)CAN4TGCA, which appears twice in the promoter
sequence, between
46 and
57 and between
67 and
78 (Fig. 1).
This study was designed to shed light on the nucleotides in the Pm
promoter that are critical for XylS-dependent stimulation of transcription.
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MATERIALS AND METHODS |
Bacterial Strains, Culture Medium, and
Plasmids--
Escherichia coli MC4100 was grown at 30 °C
in Luria-Bertani medium supplemented, when required, with 100 µg/ml
ampicillin, 25 µg/ml kanamycin, or 50 µg/ml streptomycin.
The plasmids used in this study, and previously constructed were:
pERD103, which is an IncQ plasmid encoding kanamycin resistance (7);
pJLR100, which is a pEMBL9 derivative bearing the Pm promoter cloned
between the EcoRI and HindIII sites (3); pMD1405,
which carries a promoterless 'lacZ gene and encodes
resistance to ampicillin; and pJLR107, which is a pMD1405 derivative
bearing the Pm promoter in front of 'lacZ (3).
DNA Techniques--
DNA preparation, digestion with restriction
enzymes, analysis by agarose gel electrophoresis, isolation of DNA
fragments, ligations, transformations, and sequencing reactions were
done according to standard procedures (10).
Methylation Experiments--
DNA was methylated in
vitro with 2 mM dimethyl sulfate as described (10).
For in vivo DNA metylation, E. coli cells bearing the Pm promoter in pMD1405 were exposed to 2 mM dimethyl
sulfate for 1 min at 30 °C. Cells were processed as described (10). Plasmid DNA was extracted by using the Qiagen Kit (Quiagen, GmbH, Hilden, Germany).
The oligonucleotide 5'-CGTCTAAGAAACCATTATTATCAG-3' was complementary to
the noncoding strand upstream from the Pm promoter region. The first C
at the 5'-end was located 218 bp from the +1 of the transcription
initiation point. The oligonucleotide 5'-GGGTCGGTGAACATCTCGCGCTTGC-3'
was complementary to the coding strand downstream from the Pm promoter.
The first G at the 5'-end was located 124 bp from the +1 of the start
of the transcript.
For primer extension with Taq-DNA-polymerase, about 2 × 105 cpm of the corresponding oligonucleotide end-labeled
with 32P was used. The extension products were separated by
electrophoresis on urea-polyacrylamide sequencing gels.
Construction of Pm Mutant Promoters by Polymerase Chain
Reaction--
The Pm mutant promoters were generated by overlap
extension polymerase chain reaction mutagenesis as described (5). The internal oligonucleotide primers used for mutagenesis exhibited one or
more mismatches with respect to the wild-type sequence. The external
oligonucleotide was the so-called M13 reverse primer (5'-CAGGAAACAGCTATGACCATG-3') or a primer complementary to the
fragment of the lacZ gene (5'-GATGTGCTGCAAGGCGATTAAGTTA-3'). After DNA amplification, the resulting DNA was digested with
EcoRI and HindIII, and the 401-bp
EcoRI-HindIII Pm mutants were inserted between
the EcoRI-HindIII sites of pMD1405 to yield plasmids pMARx (the x indicates the plasmid number from 5D to 296). All the mutant Pm
promoters generated in this study were confirmed by DNA sequencing.
-Galactosidase Assays--
E. coli bearing the
wild-type Pm::'lacZ or mutant
Pm*::'lacZ fusions in pMD1405, plus pERD103, were
grown overnight on Luria-Bertani medium containing the appropriate
antibiotics. Cultures were diluted 100-fold in triplicate in the same
medium supplemented or not with 1 mM 3MB. After 4 h of
incubation,
-galactosidase activity was determined in duplicate in
permeabilized cells (5).
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RESULTS |
In Vivo Methylation Assays Located the XylS Binding Region Adjacent
to the RNA Polymerase Binding Site--
The reactivity of guanine
residues in the Pm promoter toward dimethyl sulfate was assayed
in vivo. DNA protection analysis was done in E. coli bearing only pJLR107 (Pm) or pJLR107 and pERD103 (XylS) and
in the absence and in the presence of 3MB. Methylation was done when
cells had reached the mid-logarithmic growth phase. As a control, Pm
was also methylated in vitro. Representative results are
shown in Fig. 2. In the bottom strand, a significant feature of the
in vitro and in vivo methylation pattern is
hypermethylation of the T located at
42. This indicates that the DNA
was distorted at this point, probably due to the tracks of As in the
41 to
46 region in the top strand (Fig.
1). The methylation of T
42
in vivo in cells without XylS was more pronounced than
in vitro, indicating that this distortion may have been more
pronounced in vivo. The methylation pattern of Pm in
vivo in cells without XylS was very similar in the absence and in
the presence of 3MB. However, the presence of the effector influenced
the methylation pattern of Pm in cells bearing the XylS protein. When
cells expressed the XylS protein, methylation of T
42 was
highest in the presence of the effector, whereas in the absence of the
effector, T
42 appeared to be more protected than in cells
without XylS (Fig. 2). In contrast with
this behavior was the observation that Gs in the
47 to
61 region
appeared to be protected in the presence of the effector. However,
G
68 appeared to be more methylated in the presence of
effector (Fig. 2). This set of results suggested that the XylS protein
is able to bind to Pm; however, the critical interactions could not be deduced from this assay.

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Fig. 1.
Nucleotide sequence of the Pm promoter.
The proposed +1 nucleotide is based on reverse transcriptase extension
of primers that overlap the mRNA synthesized from this promoter (2,
31). The two arrows over the 70 to 35 region are
nucleotides important for XylS recognition in Pm, as proposed by
Kessler et al. (6); the two dashed arrows at the
bottom represent the motif proposed by Gallegos et
al. (5).
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Fig. 2.
In vivo footprinting of XylS.
E. coli MC4100 cells bearing only plasmid pJLR107
(Pm::'lacZ) or this plasmid plus pERD103
(xylS) were grown in the presence and in the absence of 3MB,
and then exposed to dimethyl sulfate. The panel on the left
shows the region of the bottom strand between 77 and 35, and the
panel on the right shows the region of the top strand
between 40 and +3 with respect to the main transcription initiation
point. Lane V is the same DNA methylated in
vitro. The G bases at 77, 68, 61, 56, 47, 39, 37,
24 and 3 are indicated, and the position corresponding to T at 42
is also shown.
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In the top strand in Fig. 2, the
3 to
40 region showed a definite
pattern of methylation. The Gs in the
3 to
28 region were more
methylated in vitro than in vivo. In particular,
G
23 and G
38 were clearly protected in
vivo regardless of the presence of XylS and the presence of an effector.
Block Scanning Mutagenesis of the Putative XylS Binding Site at the
Pm Promoter--
A characteristic of the members of the AraC/XylS
family of transcriptional regulators is that they recognize short
nucleotide motifs (4-6 bp) at their cognate promoters (11). For this
reason, we decided to carry out initial block scanning mutagenesis
assays of the Pm region between
41 and
78. This interval includes
all nucleotides previously proposed as important in XylS for Pm
recognition (5-7) and the region in which in vivo
footprinting analyses revealed alterations in the methylation pattern.
The interval excluded mutations in the putative
35 region of the Pm
(Fig. 1), which, as shown in the in vivo footprinting
analysis, may interfere with recognition of the promoter by the RNA
polymerase. A series of 4-bp blocks to induce mutations were introduced
randomly by polymerase chain reaction. These included the four TNCA
motifs found in this region (
46/
49,
54/
57,
67/
70, and
75/
78), the intervening sequences between two adjacent TNCA
sequences (
50/
53,
58/
61,
62/
65,
63/
66, and
71/
74),
a sequence between the TNCA sequence closer to the RNA polymerase
binding site (
46/
49), and the
41 nucleotide (
41/
44). In all
cases the mutant promoters (Pm*) were fused to
'lacZ in pMD1405.
-Galactosidase activity was determined
in E. coli MC4100
(Pm*::'lacZ, pERD103) grown in the
absence and in the presence of 3MB. The basal level of expression from
the Pm* promoters (50-100 Miller units) was similar to the basal level
of expression determined from the Pm wild-type promoter. However, with
regard to the induced levels of expression of the mutant promoters,
three classes of Pm mutants were found (Table
I): 1) mutant promoters that exhibited less than 20% of the wild-type activity; 2) mutant Pm promoters with a
level of XylS-dependent expression between 65 and 25% of the expression measured with the wild-type promoter; and 3) mutant Pm
promoters that conserved wild-type or near wild-type
XylS-dependent inducible
-galactosidase activity,
i.e.
80% of wild-type levels.
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Table I
Block scanning mutagenesis of the upstream region of the Pm promoter
and its effect on its transcriptional activity
E. coli MC4100 bearing the indicated Pm*:'lacZ
fusion and xylS in pERD103 were grown on LB medium with 3MB
as described under "Materials and Methods." In the absence of 3MB,
basal activity was 50-150 Miller units. The data correspond to induced
levels and are averages of at least six independent determinations,
with standard deviations below 20% of the given values.
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The mutations that resulted in the largest reduction in transcription,
i.e. a decrease equal to or greater than 80% of the wild-type activity, were any random substitution of the TGCA sequence between
46/
49 (Table I). This suggests that these nucleotides are
critical for XylS-dependent transcription activation of Pm. The substitution by random sequences of the AAAAA sequence located at
41/
45, the TACA sequence between
54/
57, the CGGA sequence between
58/
61, and the TGCA sequence between
67/
70 resulted in
a significant decrease in XylS-dependent transcription
activation of Pm. The activity of most of the mutant Pm promoters at
these locations ranged from approximately 25 to about 65% of the
activity of the wild-type promoter (Table I), although certain
substitutions had little effect. These results suggest that these sets
of bases are less critical than those at the
46/
49 region; however,
they may play a direct role in the recognition of the Pm DNA sequences by XylS, or they may contribute to the overall affinity for Pm. We
cannot rule out other effects.
The third group of mutations, i.e. those that had no effect
(or little effect) on transcription from Pm, were found to correspond to the locations of
50/
53,
62/
65,
63/
66,
71/
74, and
75/
78.
We also investigated whether the combination of different blocks of
mutations had a synergistic effect on XylS-dependent
transcription activation from the mutant promoters. The combination of
a block of mutations in
46/
49 (TGCA
AGGA) with a block of
mutations at
54/
57 (TACA
TTGG) resulted in a mutant Pm promoter
(Pm 245) that had no activity at all (Table I).
At the
54/
57 block some substitutions had little effect on
XylS-dependent transcription from Pm (i.e. Pm
213, 11% reduction), whereas other substitutions at the same block had
a clear effect (i.e. Pm 212, 78% reduction). In Pm 244, we
combined the block of mutations in Pm 213 with a mutant block that had
a moderate effect on the level of expression from Pm (i.e.
67/
70 (TGCA
ACGT)). This combination had cumulative effects
(Table I). These results confirmed the essential role of the
nucleotides at these positions in XylS-dependent
transcription activation from Pm.
The transcription initiation point of the mRNA generated from a
number of the above Pm mutants promoters was the same that that
determined for the wild-type promoter (not shown). This suggests that
the mutations analyzed affected the strength of transcription from the
mutant promoters.
Single Point Mutations within the Set of Blocks That Showed or Did
Not Show Any Effect on Transcription Activation from Pm--
The above
series of assays suggested critical, less critical, and irrelevant
blocks for the XylS-dependent transcription activation from
Pm. We expected that single mutations in irrelevant sets of sequences
would have no effect at all on transcriptional activity from Pm,
whereas single mutations in the critical and important blocks of
sequences would have an effect. A number of bases at the noncritical
region were selected to introduce single point mutations:
A
51
G, A
64
C, G
65
A, A
75
C, C
77
G, and
T
78
G. These mutations, as expected, had little or no
effect (<20%) on XylS-dependent transcription from Pm
(not shown).
Of the less critical boxes (
41/
44,
54/
57,
58/
61, and
67/
70), some of the substitutions (for example, A
42
T, A
44
G, A
54
G,
A
54
T, G
69
C, and G
69
T) did not significantly affect activity from Pm (Table
II); others had an intermediate effect,
reducing the induced XylS-dependent activation of Pm by
20-50%. This effect was observed for the changes C
55
A, C
55
G, A
67
C, and
A
67
T (Table II). Within these sequences, the change
T
70
G resulted in loss of almost 90% of the activity
(Table II).
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Table II
Single point mutations at the Pm promoter
Conditions were as in the legend for Table I except that the mutant Pm
promoters were those indicated below.
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In the
58/
61 box, the G
59
C, G
60
T, and G
60
A changes had a significant effect on
transcription, as shown by the finding that
-galactosidase activity
was less than 20% of that seen with the wild-type Pm promoter. A
surprising finding was that the G
62
C change resulted
in a mutant Pm promoter that lacked activity.
In the critical
46/
49 set of bases, single bp substitutions had a
significant effect on activity. The changes A
46
T,
C
47
G or C
47
T, G
48
C, and T
49
G resulted in a 60-85% decrease in
activity. However, the change G
48
A had little or no
effect on transcriptional activity (Table II).
Role of the Bases at
54/
57--
When we compared the critical
sequences proposed by Kessler et al. (6) and Gallegos
et al. (5), we found that they had the two TGCA submotifs at
46/
49 and
67/
70 in common (Fig. 1). The hypothesis of Gallegos
et al. (5) suggested that the TACA submotif between
54/
57 was critical for transcription activation from Pm, whereas
Kessler et al. (6) suggested that only the
57/
56
nucleotides were of importance (Fig. 1). Our results with single point
mutations in the
54 to
57 region showed some effect in some
positions, but in no case was the effect large enough to fully impede
transcription (Table II). To elucidate the possible role of these four
nucleotides, we generated the set of changes involving the
54/
55,
55/
56,
56/
57, and
54/
57 positions. We found that the
54/
55 changes (CA
AC or CA
GT) did not influenced the level
of
-galactosidase activity from the mutant Pm promoters. In
contrast, the
57/
56 TA
AT change almost completely prevented
activity (99% decrease). The combination of mutations at
55/
56 and
54/
57 had an intermediate effect, with activities in the range of
15-38% when the mutation involved the
57 position, and in the range
of 40-80% when the changed involved the
56 position (not shown).
From these results, we deduced that the
57/
56 bases are critical
for the activity of Pm.
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DISCUSSION |
The XylS protein (12-14) belongs to the AraC/XylS family of
regulators that comprises more than 100 different proteins involved in
transcription stimulation of several cell processes, such as carbon
metabolism, pathogenesis, and response to alkylating agents in bacteria
(11). Members of the family for which in vitro or in
vivo footprinting assays are available (AraC, RhaR, RhaS, MelR, SoxS, and Ada) have at least two features in common: they function in vivo as a dimer, and the stretch of nucleotides covered
by a monomer of the regulatory protein at the regulated promoter is
between 15 and 20 bp long. However, within this set of bases, short
motifs seem to confer critical base recognition for DNA-protein interactions (15-21). This seems also to be the case for Pm/XylS interactions. Our in vivo footprinting analysis and the
analysis of transcriptional activity from wild-type and mutant Pm
promoters showed that critical nucleotides extended from the
70
position in the 5'-end to at least the
41 position in the 3' end. Our mutational analysis revealed that within this stretch the XylS recognition sequence seems to be TGCAN6GGNTA, which appears
twice in the Pm promoter, between
70 and
56 and between
49 and
35 (Fig. 1). In favor of this proposal is the observation that a tagged XylS-protein immunoadsorbed onto glass beads produced footprints in vitro, which showed protection of the Gs within the above
direct repeat at
48,
59,
60, and
69 (22). This is in agreement with our in vivo results.
The AraC protein
the best characterized regulator of the
family
stimulates transcription as a dimer (23-25). The consensus sequence for AraC-activable promoters is a direct repetition of the
TAGCN7TCCATA motif: each AraC monomer recognizes one of the direct repeats (26-29).
At the C-terminal end, the regulators of the AraC/XylS family show a
highly conserved stretch of about 100 amino acids that seems to be
involved in DNA binding and probably in interactions with RNA
polymerase (11). One characteristic of members of this family is that
they exhibit two possible HTH DNA binding motifs (located at 228-251
and 281-305 in XylS and at 198-217 and 246-264 in AraC). Brunelle
and Schleif (15) analyzed these possible HTH motifs with substitutions
of several amino acids that should contact DNA in an HTH structure and
found evidence for the first one. Niland et al. (30), using
synthetic oligonucleotides, systematically substituted the bases in the
AraC recognition sequence and did gel retardation assays with mutant
AraC in each of the possible HTH elements. They showed that the mutant
AraC in each of the HTH motifs exhibited altered DNA binding
properties. On the basis of their results, these authors proposed that
each AraC monomer binds the 5'-TAGC submotif with one of the HTH
motifs, and the 3'-TCCATA submotif with the second HTH.
If XylS contacts DNA via the two possible HTH elements, all mutant Pm
promoters generated in different laboratories can be explained by the
following model (Fig. 3): the XylS
protein recognizes two submotifs in Pm, TGCA and GGNTA, which are
separated from each other by six nucleotides. Each submotif is
recognized by the recognition helix of one of the HTH elements of XylS.
For the wild-type protein, recognition of direct sequences leads to the
formation of a dimer. One monomer recognizes the upstream motif (from
70 to
56) with the two HTH DNA binding elements; the second monomer
recognizes mainly the TGCA submotif and interacts with the downstream
sequence, where it may compete for binding with the RNA-polymerase
(Fig. 3). Mutations at the
46/
49 TGCA submotif result in mutants in
which the capacity to activate transcription is impaired because they
cannot be contacted properly by XylS. Failure of one of the XylS
monomers to interact with this submotif prevents dimer formation and
leads to a nonactivable mutant Pm promoter. Pm mutants at the distal
TGCA (
67/
70) submotif can be activated weakly by the wild-type XylS
protein as a result of the formation of an unstable dimer; however,
they can still be induced to a high level of activation by mutant XylS
proteins with higher affinity for target sequences than the wild-type
regulator (5). This is because one of the XylS mutant monomers binds to
the downstream motif (
49/
35); and because of the mutation in the
XylS, the second monomer is still able to interact well with the GGNTA
submotif, and this suffices for dimer formation. The transcriptional
activity of mutant Pm promoters with altered GGNTA at
60/
56
sequences is seriously impaired, because failure of one of the monomers
to bind correctly prevents dimer stabilization at Pm.